[Name of Document] APPLICATION FOR PATENT
[Identification Number] C1-A0320Y1
[Filing Date] March 12, 2004
[Addressee] Esq. Commissioner of the Patent Office
[Inventor]
[Address] c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
153-2, Nagai, Niihari-mura, Niihari-gun,
Ibaraki
TSUNODA, Hiroyuki
[Name]
[inventor]
[Address]
[ Name ]
[ Inventor]
[Address]
[ Name ]
[inventor]
[Address]
[Name]
[Inventor]
[Address]
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
153-2, Nagai, Niihari-mura, Niihari-gun,
Ibaraki
NAKANO, Kiyotaka
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA.
153-2, Nagai, Niihari-mura, Niihari-gun,
Ibaraki
ORITA, Tetsuro
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
135, Komakado 1-chome, Gotenba-shi,
Shizuoka
TSUCHIYA, Masayuki
[ Name ]
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA,
153-2, Nagai, Niihari-mura, Niihari-gun,
IBARAKI
HIRATA, Yuichi
[Applicant]
[Identification Number] 000003311
[Name or Appellation]
[Attorney]
CHUGAI SEIYAKU KABUSHIKI KAISHA
[Identification Number] 100102978
[Patent Attorney]
[Name or Appellation] Hatsushi Shimizu
[Nominated Attorney]
[Identification Number] 100108774
[Patent Attorney]
[Name or Appellation] Kazunori Hashimoto
[Priority]
[Application Number]
[Filing Date]
[ Payment]
[Registration Number]
JP2003-415746
December 12, 2003
041092
[Amount] 21000 yen
[List of Attached Documents]
[Name of Document] Claims
1
[Name of Document] Specification 1
[Name of Document] Drawings
[Name of Document] Abstract
1
1
[General Power of Attorney Number] 0216136
1
JP 2004-071763
[Document Name] Claims
[Claim 1]
An antibody comprising a single-chain polypeptide having binding activity against TPO
receptor (Mpl), wherein said antibody comprises two heavy chain variable regions and two light
5 chain variable regions.
[Claim 2]
The antibody of claim 1 , wherein the two heavy chain variable regions and the two light
chain variable regions are arranged in the order of heavy chain variable region, light chain
variable region, heavy chain variable region, and light chain variable region from the N terminus
10 of the single-chain polypeptide.
[Claim 3]
The antibody of claim 1 or 2, wherein the two heavy chain variable regions and the two
light chain variable regions are linked by linkers.
[Claim 4]
15 The antibody of claim 3, wherein the linkers comprise 15 amino acids.
[Claim 5]
A chimeric antibody that binds to Mpl.
[Claim 6]
The antibody of claim 5, which is a humanized antibody.
20 [Claim 7]
The antibody of claim 5 or 6, which is a minibody.
[Claim 8]
An antibody that binds to soluble Mpl.
[Claim 9]
25 An antibody that binds to human Mpl and monkey Mpl.
[Claim 10]
An antibody having agonistic activity against human Mpl and monkey Mpl.
[Claim 11]
An antibody whose binding activity to soluble Mpl is KD = 1 0 M or lower.
30 [Claim 12]
An antibody whose binding activity to soluble Mpl is KD = 10" 7 M or lower.
[Claim 13]
Q
An antibody whose binding activity to soluble Mpl is KD =10' M or lower.
[Claim 14]
35 An antibody whose TPO agonistic activity is EC50 = 100 nM or lower.
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[Claim 15]
An antibody whose TPO agonistic activity is EC50 = 30 nM or lower.
[Claim 16]
An antibody whose TPO agonistic activity is EC50 =10 nM or lower.
[Claim 17]
An antibody which comprises a heavy chain variable region, wherein said heavy chain
variable regions comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence of
any one of:
[I] SEQIDNOs: 3, 4, and 5
[2] SEQ ID NOs: 6, 7, and 8
[3] SEQ ID NOs: 9, 1 0, and 1 1
[4] SEQ ID NOs: 12, 13, and 14
[5] SEQ ID NOs: 15, 16, and 17
[6] SEQ ID NOs: 1 8, 1 9, and 20
[7] SEQ ID NOs: 21, 22, and 23
[8] SEQ ID NOs: 24, 25, and 26
[9] SEQ ID NOs: 27, 28, and 29
[10] SEQ ID NOs: 30, 31, and 32
[II] SEQ ID NOs: 33, 34, and 35
[12] SEQ ID NOs: 36, 37, and 38
[13] SEQ ID NOs: 39, 40, and 41
[14] SEQ ID NOs: 42, 43, and 44
[15] SEQ ID NOs: 45, 46, and 47
[16] SEQ ID NOs: 48, 49, and 50
[17] SEQ ID NOs: 51, 52, and 53
[18] SEQ ID NOs: 54, 55, and 56
[19] SEQ ID NOs: 57, 58, and 59
[20] SEQ ID NOs: 231, 233, and 235
[21] SEQ ID NOs: 266, 268, and 270
[22] SEQ ID NOs: 280, 282, and 284.
[Claim 1 8]
An antibody which comprises a light chain variable region, wherein said light chain
variable region comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence of any
one of:
[1] SEQ ED NOs: 60, 61, and 62
[2] SEQ ID NOs: 63, 64, and 65
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[3] SEQ ID NOs: 66, 67, and 68
[4] SEQ ID NOs: 69, 70, and 71
[5] SEQ ID NOs: 72, 73, and 74
[6] SEQ ID NOs: 75, 76, and 77
5 [7] SEQ ID NOs: 78, 79, and 80
[8] SEQ ID NOs: 81, 82, and 83
[9] SEQ ID NOs: 84, 85, and 86
[10] SEQ ID NOs: 87, 88, and 89
[11] SEQ ID NOs: 90, 91, and 92
10 [12] SEQ ID NOs: 93, 94, and 95
[13] SEQ ID NOs: 96, 97, and 98
[14] SEQ ID NOs: 99, 100, and 101
[15] SEQ ID NOs: 102, 103, and 104
[16] SEQ ID NOs: 105, 106, and 107
15 [17] SEQ ID NOs: 108, 109, and 110
[18] SEQ ID NOs: 111, 112, and 113
[19] SEQ ID NOs: 114, 115, and 116
[20] SEQ ID NOs: 240, 242, and 244
[21] SEQ ID NOs: 273, 275, and 277.
20 [Claim 19]
An antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
[1] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino
acid sequences consisting of SEQ ID NOs: 3, 4, and 5, and a light chain variable region that
25 comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 60, 61, and 62;
[2] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino
acid sequences consisting of SEQ ID NOs: 6, 7, and 8, and a light chain variable region that
comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting of SEQ ID
30 NOs: 63, 64, and 65;
[3] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino
acid sequences consisting of SEQ ID NOs: 9, 10, and 1 1 , and a light chain variable region that
comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 66, 67, and 68;
35 [4] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising the amino
acid sequences consisting of SEQ ID NOs: 12, 13, and 14, and a light chain variable region that
JP 2004-071763
comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting of SEQ ID
NOs: 69, 70, and 71;
[5] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 15, 16, and 17, and a light chain variable
5 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 72, 73, and 74;
[6] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 18, 19, and 20, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
10 of SEQ ID NOs: 75, 76, and 77;
[7] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 21, 22, and 23, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;
15 [8] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 24, 25, and 26, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 81, 82, and 83;
[9] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
20 the amino acid sequences consisting of SEQ ID NOs: 27, 28, and 29, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 84, 85, and 86;
[10] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 30, 31, and 32, and a light chain variable
25 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 87, 88, and 89;
[11] a heavy chain variable region that comprises CDR1, CDR2 5 and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 33, 34, and 35, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
30 of SEQ ID NOs: 90, 91, and 92;
[12] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 36, 37, and 38, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 93, 94, and 95;
35 [13] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 39, 40, and 41, and a light chain variable
5 JP 2004-071763
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 96, 97, and 98;
[14] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 42 3 43, and 44, and a light chain variable
5 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 99, 100, and 101;
[15] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 45, 46, and 47, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
10 of SEQ ID NOs: 102, 103, and 104;
[16] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 48, 49, and 50, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 105, 106, and 107;
15 [17] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 51, 52, and 53, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
ofSEQIDNOs: 108, 109, and 110,
[18] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
20 the amino acid sequences consisting of SEQ ID NOs: 54, 55, and 56, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
ofSEQIDNOs: 111, 112, and 113;
[19] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 57, 58, and 59, and a light chain variable
25 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
ofSEQIDNOs: 114, 115, and 116;
[20] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 23 1, 233, and 235, and a light chain
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
30 consisting of SEQ ID NOs: 240, 242, and 244;
[21] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 266, 268, and 270, and a light chain
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
consisting of SEQ ID NOs: 273, 275, and 277;
35 [22] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 280, 282, and 284, and a light chain
6 JP 2004-071763
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
consisting of SEQ ID NOs: 273, 275, and 277.
[Claim 20]
An antibody that comprises a heavy chain variable region comprising the amino acid
5 sequence of SEQ ID NO: 1 1 8.
[Claim 21]
An antibody that comprises a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 120.
[Claim 22]
1 0 An antibody that comprises a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 118 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 120.
[Claim 23]
An antibody comprising the amino acid sequence of SEQ ID NO: 122 or 264.
15 [Claim 24]
An antibody that comprises a heavy chain variable region, wherein said heavy chain
variable region comprises FR1, FR2, FR3, and FR4 consisting of amino acid sequences of any
one of:
[1] SEQ ID NOs: 230, 232, 234, and 236
20 [2] SEQ ID NOs: 265, 267, 269, and 271
[3] SEQ ID NOs: 279, 281, 283, and 285.
[Claim 25]
An antibody comprising a light chain variable region, wherein said light chain variable
region comprises FR1, FR2, FR3, and FR4 consisting of amino acid sequences of any one of:
25 [1] SEQ ID NOs: 239, 241, 243, and 245
[2] SEQ ID NOs: 272, 274, 276, and 278.
[Claim 26]
An antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
30 [1] a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having the
amino acid sequences consisting of SEQ ID NOs: 230, 232, 234, and 236, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 239, 241, 243, and 245;
[2] a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having the
35 amino acid sequences consisting of SEQ ID NOs: 265, 267, 269, and 271, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
7
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consisting of SEQ ID NOs: 272, 274, 276, and 278;
[3] a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having the
amino acid sequences consisting of SEQ ID NOs: 279, 281, 283, and 285, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278.
[Claim 27]
An antibody that comprises a heavy chain variable region, wherein said heavy chain
variable region comprises the amino acid sequence of SEQ ID NO: 229, 256, or 262.
[Claim 28]
An antibody that comprises a light chain variable region, wherein said light chain
variable region comprises the amino acid sequence of SEQ ID NO: 238 or 258.
[Claim 29]
An antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
[1] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
229, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 238;
[2] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
256, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258;
[3] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
262, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258.
[Claim 30]
An antibody that comprises the amino acid sequence of SEQ ID NO: 2, 254, or 260.
[Claim 31]
An antibody having an activity equivalent to that of an antibody of any one of claims 1 7
to 30, wherein said antibody comprises the amino acid sequence set forth in any one of claims 17
to 30, in which one or more amino acids have been substituted, deleted, added and/or inserted.
[Claim 32]
An antibody that recognizes an epitope recognized by an antibody of any one of claims
17to31.
[Claim 33]
An antibody that recognizes the region of amino acids 26 to 274 of human Mpl.
[Claim 34]
An antibody of any one of claims 1 to 33, which has TPO agonistic activity.
[Claim 35]
A polynucleotide encoding an antibody of any one of claims 1 to 34.
[Claim 36]
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A polynucleotide hybridizing to the polynucleotide of claim 35 under stringent
conditions, wherein said polynucleotide encodes an antibody having activity equivalent to that of
an antibody of any one of claims 1 to 34.
[Claim 37]
5 A vector comprising the polynucleotide of claim 35 or 36.
[Claim 38]
A host cell that carries the polynucleotide of claim 35 or 36, or the vector of claim 37.
[Claim 39]
A pharmaceutical composition comprising an antibody of any one of claims 1 to 34.
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[Document Name] Specification
[Title of the Invention] ANTI-MPL ANTIBODIES
[Technical Field]
[0001]
The present invention relates to anti-Mpl antibodies.
[Background of the Invention]
[0002]
Thrombopoietin (TPO) is a factor that enhances the differentiation and maturation of
megakaryocytes (platelet precursor cells) from hemopoietic stem cells into platelets. TPO also
functions as a cytokine with an important role in the regulation of platelet number. TPO is
converted into its active form through the cleavage of a TPO precursor comprising 353 amino
acids.
[0003]
Mpl is a TPO receptor, and human Mpl molecules are known to exist in two forms
comprising 572 and 635 amino acids. The human Mpl gene sequence has already been
analyzed (see Non-patent Document 1 and GenBank accession No. NM_005373).
[0004]
Most cytokine receptors dimerize upon ligand binding, and transduce signals into cells.
It has been reported that TPO similarly binds to its own specific receptor MPL, which leads to
dimerization of the receptor, thereby transducing signals into cells and exerting physiological
effects (see Non-patent Document 2).
[0005]
Antibodies exhibiting agonistic activity have been reported among those antibodies that
bind to receptors having the above features.
For example, an antibody against the erythropoietin (EPO) receptor has been reported to
substitute for erythropoietin function. The monovalent form (Fab) of the antibody is capable of
binding to the EPO receptor but is unable to transduce signals. Thus, dimerization of the
erythropoietin receptor via bivalent binding is assumed to be essential for signal transduction
(see Non-patent Document 3).
[0006]
Antibodies that bind to Mpl and exhibit TPO agonistic activity have also been reported
(see Non-patent Documents 4 and 5). This suggests that receptor dimerization is induced upon
binding of a bivalent antibody with regards to MPL as well.
[0007]
Meanwhile, a single-chain antibody (scFv) has been reported to exhibit TPO agonistic
JP 2004-071763
activity (see Patent Document 1). However, it has been revealed that, the underlying
mechanism of scFv exhibiting TPO agonistic activity is that a part of scFv dimerizes (diabody)
and this diabody becomes the actual active unit (see Patent Documents 2 to 4).
[0008]
5 [Patent Document 1] US Patent No. 6342220
[Patent Document 2] WO 01/79494
[Patent Document 3] WO 02/33072
[Patent Document 4] WO 02/33073
[Non-patent Document 1] Palacios et al, 1985, Cell, 41, 727-734
10 [Non-patent Document 2] Souyri et al, 1990, Cell, Vol.63, 1137-1147
[Non-patent Document 3]Elliott, S. et al, 1996, J. Biol. Chem., 271(40), 24691-24697
[Non-patent Document 4] Abe et al, 1998, Immunol. Lett., 61, 73-78
[Non-patent Document 5] Bijia Deng et al., 1998, Blood, 92, 1981-1988
[Disclosure of the Invention]
1 5 [Problems to be Solved by the Invention]
[0009]
The present invention was achieved in view of the above circumstances. An objective
of the present invention is to provide novel anti-Mpl antibodies having TPO agonistic activity.
[Means for Solving the Problems]
20 [0010]
The present inventors performed exhaustive research to solve the above objective. The
present inventors prepared and purified anti-human Mpl antibody VB22B, and established a
single-chain antibody expression system using genetic engineering techniques. Specifically, the
variable region of anti-human Mpl antibody was first cloned, and a diabody expression vector
25 pCXND3-VB22B db for the anti-human Mpl antibody was prepared. This pCXND3-VB22B
db vector was then used to generate an expression vector pCXND3-VB22B sc(Fv) 2 for
anti-human Mpl antibody sc(Fv) 2 . Anti-human Mpl sc(Fv) 2 was expressed in CHO-DG44 cells
using the expression vector pCXND3-VB22B sc(Fy) 2 , and then purified from the culture
supernatant. In control experiments, VB22B diabody was transiently expressed in COS7 cells
30 using the above pCXND3-VB22B db vector, and then purified from the culture supernatant.
[0011]
In addition, VB22B diabody and VB22B sc(Fv) 2 were evaluated for their TPO-like
agonistic activities. The results showed that VB22B diabody and VB22B sc(Fv) 2 exhibit higher
agonistic activities compared to VB22B IgG, and thus activities equivalent to or higher than that
35 of the natural ligand, human TPO.
[0012]
11
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Furthermore, the present inventors succeeded in preparing three types of humanized
VB22B sc(Fv)2. The TPO-like agonistic activity was also proven to be unaltered by
humanization.
[0013]
More specifically, the present invention relates to:
(1) an antibody comprising a single-chain polypeptide having binding activity against
TPO receptor (Mpl), wherein said antibody comprises two heavy chain variable regions and two
light chain variable regions;
(2) the antibody of (1), wherein the two heavy chain variable regions and the two light
chain variable regions are arranged in the order of heavy chain variable region, light chain
variable region, heavy chain variable region, and light chain variable region from the N terminus
of the single-chain polypeptide;
(3) the antibody of (1) or (2), wherein the two heavy chain variable regions and the two
light chain variable regions are linked by linkers;
(4) the antibody of (3), wherein the linkers comprise 15 amino acids;
(5) a chimeric antibody that binds to Mpl;
(6) the antibody of (5), which is a humanized antibody;
(7) the antibody of (5) or (6), which is a minibody;
(8) an antibody that binds to soluble Mpl;
(9) an antibody that binds to human Mpl and monkey Mpl;
(10) an antibody having agonistic activity against human Mpl and monkey Mpl;
(11) an antibody whose binding activity to soluble Mpl is KD = 10" 6 M or lower;
(12) an antibody whose binding activity to soluble Mpl is KD = 10' 7 M or lower;
(13) an antibody whose binding activity to soluble Mpl is KD = 10" 8 M or lower;
(14) an antibody whose TPO agonistic activity is EC50 = 100 nM or lower;
(15) an antibody whose TPO agonistic activity is EC50 = 30 nM or lower;
(16) an antibody whose TPO agonistic activity is EC50 =10 nM or lower;
(17) an antibody which comprises a heavy chain variable region, wherein said heavy
chain variable regions comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence
of any one of:
[1] SEQIDNOs: 3,4, and 5
[2] SEQ ID NOs: 6, 7, and 8
[3] SEQ ID NOs: 9, 10, and 11
[4] SEQ ID NOs: 12, 13, and 14
[5] SEQ ID NOs: 15, 16, and 17
[6] SEQ ID NOs: 18, 19, and 20
12
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7] SEQ ID NOs: 21, 22, and 23
8] SEQ ID NOs: 24, 25, and 26
9] SEQ ID NOs: 27, 28, and 29
10] SEQ ID NOs: 30, 31, and 32
11] SEQ ID NOs: 33, 34, and 35
12] SEQ ID NOs: 36, 37, and 38
13] SEQ ID NOs: 39, 40, and 41
14] SEQ ID NOs: 42, 43, and 44
15] SEQ ID NOs: 45, 46, and 47
10 T16] SEQ ID NOs: 48, 49, and 50
17] SEQ ID NOs: 51, 52, and 53
1 8] SEQ ID NOs: 54, 55, and 56
19] SEQ ID NOs: 57, 58, and 59
20] SEQ ID NOs: 231, 233, and 235
15 T2 1 ] SEQ ID NOs: 266, 268, and 270
22] SEQ ID NOs: 280, 282, and 284;
1 8) an antibody which comprises a light chain variable region, wherein said light chain
variable region comprises CDR1, CDR2 and CDR3 consisting of an amino acid sequence of any
one of:
20 [1] SEQ ID NOs: 60, 61, and 62
[2] SEQ ID NOs: 63, 64, and 65
[3] SEQ ID NOs: 66, 67, and 68
[4] SEQ ID NOs: 69, 70, and 71
[5] SEQ ID NOs: 72, 73, and 74
25 [6] SEQ ID NOs: 75, 76, and 77
[7] SEQ ID NOs: 78, 79, and 80
[8] SEQ ID NOs: 81, 82, and 83
[9] SEQ ID NOs: 84, 85, and 86
[10] SEQ ID NOs: 87, 88, and 89
30 [11] SEQ ID NOs: 90, 91, and 92
[12] SEQ ID NOs: 93, 94, and 95
[13] SEQ ID NOs: 96, 97, and 98
[14] SEQ ID NOs: 99, 100, and 101
[15] SEQ ED NOs: 102, 103, and 104
35 [16] SEQ ID NOs: 105, 106, and 107
[17] SEQ ID NOs: 108, 109, and 110
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[18] SEQIDNOs: 111, 112, and 113
[19] SEQ IDNOs: 114, 115, and 116
[20] SEQ ID NOs: 240, 242, and 244
[21] SEQ ID NOs: 273, 275, and 277;
5 (19) an antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
[1] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 3, 4, and 5, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
1 0 of SEQ ID NOs: 60, 6 1 , and 62;
[2] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 6, 7, and 8, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 63, 64, and 65;
15 [3] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 9, 10, and 11, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 66, 67, and 68;
[4] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
20 the amino acid sequences consisting of SEQ ID NOs: 12, 13, and 14, and a light chain variable
region that comprises CDR1 , CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 69, 70, and 71;
[5] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 15, 16, and 17, and a light chain variable
25 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 72, 73, and 74;
[6] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 18, 19, and 20, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
30 of SEQ ID NOs: 75, 76, and 77;
[7] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 21, 22, and 23, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 78, 79, and 80;
35 [8] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 24, 25, and 26, and a light chain variable
7
14 JP 2004-071763
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 81, 82, and 83;
[9] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 27, 28, and 29, and a light chain variable
5 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 84, 85, and 86;
[ 1 0] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 30, 31, and 32, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
10 ofSEQIDNOs: 87, 88, and 89;
[1 1] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 33, 34, and 35, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 90, 91, and 92;
15 [12] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 36, 37, and 38, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 93, 94, and 95;
[13] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
20 the amino acid sequences consisting of SEQ ID NOs: 39, 40, and 41, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 96, 97, and 98;
[14] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 42, 43, and 44, and a light chain variable
25 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
ofSEQIDNOs: 99, 100, and 101;
[15] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 45, 46, and 47, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
30 ofSEQIDNOs: 102, 103, and 104;
[16] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 48, 49, and 50, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ ID NOs: 105, 106, and 107;
35 [17] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 51, 52, and 53, and a light chain variable
15 JP 2004-071763
region that comprises CDR1, CDR2 5 and CDR3 comprising the amino acid sequences consisting
ofSEQ IDNOs: 108, 109, and 110;
[18] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 54, 55, and 56, and a light chain variable
5 region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
of SEQ IDNOs: 111, 112, and 113;
[19] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 57, 58, and 59, and a light chain variable
region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences consisting
10 of SEQ ID NOs: 114, 115, and 116;
[20] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 231, 233, and 235, and a light chain
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
consisting of SEQ ID NOs: 240, 242, and 244;
15 [2 1 ] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
the amino acid sequences consisting of SEQ ID NOs: 266, 268, and 270, and a light chain
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
consisting of SEQ ID NOs: 273, 275, and 277;
[22] a heavy chain variable region that comprises CDR1, CDR2, and CDR3 comprising
20 the amino acid sequences consisting of SEQ ID NOs: 280, 282, and 284, and a light chain
variable region that comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences
consisting of SEQ ID NOs: 273, 275, and 277;
(20) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 118;
25 (21) an antibody that comprises a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 120;
(22) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 118 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 120;
30 (23) an antibody comprising the amino acid sequence of SEQ ID NO: 122 or 264;
(24) an antibody that comprises a heavy chain variable region, wherein said heavy chain
variable region comprises FR1, FR2, FR3, and FR4 consisting of amino acid sequences of any
one of:
[1] SEQ ID NOs: 230, 232, 234, and 236
35 [2] SEQ ID NOs: 265, 267, 269, and 271
[3] SEQ ID NOs: 279, 281, 283, and 285.
16
JP 2004-071763
(25) an antibody comprising a light chain variable region, wherein said light chain
variable region comprises FR1, FR2, FR3, and FR4 consisting of amino acid sequences of any
one of:
[1] SEQ ID NOs: 239, 241, 243, and 245
5 [2] SEQ ID NOs: 272, 274, 276, and 278;
(26) an antibody that comprises a heavy chain variable region and a light chain variable
region of any one of:
[1] a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having the
amino acid sequences consisting of SEQ ID NOs: 230, 232, 234, and 236, and a light chain
10 variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 239, 241, 243, and 245;
[2] a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having the
amino acid sequences consisting of SEQ ID NOs: 265, 267, 269, and 271, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
15 consisting of SEQ ID NOs: 272, 274, 276, and 278;
[3] a heavy chain variable region which comprises FR1, FR2, FR3, and FR4 having the
amino acid sequences consisting of SEQ ID NOs: 279, 281, 283, and 285, and a light chain
variable region which comprises FR1, FR2, FR3, and FR4 having the amino acid sequences
consisting of SEQ ID NOs: 272, 274, 276, and 278;
20 (27) an antibody that comprises a heavy chain variable region, wherein said heavy chain
variable region comprises the amino acid sequence of SEQ ID NO: 229, 256, or 262;
(28) an antibody that comprises a light chain variable region, wherein said light chain
variable region comprises the amino acid sequence of SEQ ID NO: 238 or 258;
(29) an antibody that comprises a heavy chain variable region and a light chain variable
25 region of any one of:
[1] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
229, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 238;
[2] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
256, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258;
30 [3] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
262, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 258;
(30) an antibody that comprises the amino acid sequence of SEQ ID NO: 2, 254, or 260;
(3 1) an antibody having an activity equivalent to that of an antibody of any one of (1 7)
to (30), wherein said antibody comprises the amino acid sequence set forth in any one of (17) to
35 (30), in which one or more amino acids have been substituted, deleted, added and/or inserted;
(32) an antibody that recognizes an epitope recognized by an antibody of any one of
F
17 JP 2004-071763
(17) to (31);
(33) an antibody that recognizes the region of amino acids 26 to 274 of human Mpl;
(34) an antibody of any one of (1) to (33), which has TPO agonistic activity;
(35) a polynucleotide encoding an antibody of any one of (1) to (34);
5 (36) a polynucleotide hybridizing to the polynucleotide of (35) under stringent
conditions, wherein said polynucleotide encodes an antibody having activity equivalent to that of
an antibody of any one of (1) to (34);
(37) a vector comprising the polynucleotide of (35) or (36);
(38) a host cell that carries the polynucleotide of (35) or (36), or the vector of (37); and
10 (39) a pharmaceutical composition comprising an antibody of any one of (1) to (34).
[Effects of the Invention]
[0014]
Recombinant human TPOs have been tested as therapeutic agents for
chemotherapy- induced thrombocytopenia in various clinical trials. In these trials, a major
15 problem that was reported was the production of anti-TPO antibody in TPO-treated patients
(Junzhi Li, et al., Blood (2001) 98: 3241-324; Saroj Vandhan-Raj, et al., Ann. Intern. Med.
(2000) 132: 364-368). Specifically, production of neutralizing antibodies that inhibit
endogenous TPO activity, and the resulting onset of thrombocytopenia, were reported.
Administration of agonistic minibodies (low molecular weight antibodies) against anti-TPO
20 receptor of the present invention does not induce the production of antibodies against
endogenous TPO. Furthermore, since antibody miniaturization results in increased specific
activity and short half-life in blood, the effective concentration in blood can be easily regulated,
presenting a further clinical advantage. Accordingly, the present antibodies are expected to be
therapeutic agents for thrombocytopenia which are more effective than naturally-occurring TPO
25 or its agonistic antibodies. Furthermore, since minibodies are not glycosylated, expression
systems for expressing the recombinant proteins are not limited, and minibodies can be produced
in any expression system such as cell lines from mammals, yeast, insect cells, and E. coli.
Since binding specificity against mutant TPO receptor is different from that of TPO, minibodies
are expected to bind specific mutants and show agonistic activity against mutated TPO receptor
30 detected in the CAMT patients, who genetically have mutated TPO receptor and develop
thrombocytopenia.
[Best Mode for Carrying Out the Invention]
[0015]
The present invention provides antibodies that bind to the TPO receptor (Mpl).
35 [0016]
The antibodies of the present invention comprise all types of antibodies, including
18 JP 2004-071763
antibodies with modified amino acid sequences, such as minibodies, humanized antibodies, and
chimeric antibodies; antibodies that have been modified by binding with other molecules (for
example, polymers such as polyethylene glycol); and antibodies whose sugar chains have been
modified.
5 [0017]
Mpl of the present invention may be a mutant receptor. A mutant receptor of the
present invention is usually a receptor that exists at a frequency lower than 50%, preferably
lower than 20%, more preferably lower than 10%, and even more preferably lower than 1%.
The frequency is generally calculated using randomly selected subjects. However, the
1 0 frequency may vary depending on the country, area, sex, and such. Therefore, the frequency
may also be calculated, for example, within a defined country or area, such as Japan, the United
States, and Europe, or calculated for one sex. When there are two or more mutations in a
receptor, the frequency may be calculated for multiple mutation sites or for any one of the
mutation sites. Mutant receptors are preferably evaluated by a frequency as described above.
1 5 However, mutant receptors can also be evaluated, for example, by their signal transducing ability
and such. Specifically, for example, when two different receptors are present, the one with
stronger transducing signals upon natural ligand-binding maybe be used as a non-mutant type
receptor, and the one with weaker transducing signals as a mutant receptor.
[0018]
20 In one embodiment, the mutant receptors of the present invention comprise receptors
that are associated with disease onset. The phrase "mutant receptors associated with disease
onset" means that the loss of reactivity to a natural ligand becomes part of the reason that
triggers disease onset. In the present invention, the mutant receptor may be a contributing
factor, but not necessarily the sole factor triggering disease onset. Many reports have been
25 previously published that describe the association of mutant receptors with disease onset. In
addition to those that have been reported, associations of mutant receptors and disease onset can
also be identified by statistical analysis methods (for example, correlation analyses).
Correlation analyses, also called "case control studies", are well known to those skilled in the art
(for example, Nishimura, Y., 1991, "Statistical analysis of polymorphisms", Saishin Igaku,
30 46:909-923; Oka, A. et aL, Hum. Mol. Genetics (1990) 8: 2165-2170; Ota, M. et al, Am. J. Hum.
Genet. (1999) 64: 1406-1410; Ozawa, A. etai, Tissue Antigens (1999) 53: 263-268). For
example, the correlation between a mutant receptor and a disease can be studied by computing
the frequency of the mutant receptor in patients and healthy subjects, and testing whether the
patient population has a higher mutant receptor frequency. Typically, differences in frequency
35 are evaluated using the x-test. % is obtained by the equation % 2 = Z (observed value - expected
value) /expected value. A p value is obtained from the x value determined. Based on this p
JP 2004-071763
value, it can be determined whether there is a correlation between the mutant receptor and the
disease. For example, when p<0.05, the mutant receptor is considered to correlate with the
disease. Mutant thrombopoietin (TPO) receptors have already been reported (Matthias
Ballmaier et aL, BLOOD (2001) 97 (1): 139; and others).
5 [0019]
It is preferable that the antibodies of the present invention have agonistic activity against
Mpl.
In a preferred embodiment, the antibodies of the present invention comprise, for
example, minibodies.
10 The minibodies comprise antibody fragments lacking portions of the whole antibody
(for example, whole IgG). The minibodies are not particularly limited as long as they have
binding activity to their antigens. The minibodies of the present invention have markedly
higher activities compared to their corresponding whole antibodies. There are no particular
limitations on the antibody fragments of the present invention as long as they are portions of the
1 5 whole antibody, and preferably contain heavy chain variable regions (VH) and/or light chain
variable regions (VL). The amino acid sequences of VH or VL may contain substitutions,
deletions, additions and/or insertions. Furthermore, the antibody fragment may also lack
portions of VH or/and VL, as long as it has binding ability to its antigen. In addition, the
variable regions may be chimerized or humanized. Such antibody fragments include, for
20 example, Fab, Fab', F(ab')2, and Fv. An example of a minibody includes Fab, Fab', F(ab')2, Fv,
scFv (single-chain Fv), diabody, and sc(Fv)2 (single-chain (Fv) 2 ).
[0020]
Herein, an "Fv" fragment is the smallest antibody fragment and contains a complete
antigen recognition site and a binding site. The "Fv" fragment is a dimer (VH-VL dimer) in
25 which a single VH and a single VL are strongly linked by a non-covalent bond. The three
complementarity-determining regions (CDRs) of each of the variable regions interact with each
other to form an antigen-binding site on the surface of the VH-VL dimer. Six CDRs confer the
antigen-binding site of an antibody. However, a single variable region (or a half of Fv
containing only three CDRs specific to an antigen) alone is also capable of recognizing and
30 binding an antigen although its affinity is lower than the affinity of the entire binding site.
[0021]
scFv contains the VH and VL regions of an antibody, and these regions exist on a single
polypeptide chain. Generally, an Fv polypeptide further contains a polypeptide linker between
VH and VL, and therefore an scFv can form a structure required for antigen binding. See,
35 Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113 (Rosenburg and Moore eds.
(Springer Verlag, New York, pp.269-315, 1994) for the review of scFv. In the present invention,
20
JP 2004-071763
linkers are not especially limited as long as they do not inhibit expression of antibody variable
regions linked at both ends of the linkers.
[0022]
The term "diabody" refers to a bivalent antibody fragment constructed by gene fusion
5 (Holliger P et al , Proc. Natl. Acad. Sci. USA (1993) 90: 6444-6448; EP 404,097; WO 93/1 1161
and others). Diabodies are dimers comprising two polypeptide chains, where each polypeptide
chain comprises a VL and a VH connected with a linker short enough to prevent interaction of
these two domains, for example, a linker of about five residues. The VL and VH encoded on
the same polypeptide chain will form a dimer because the linker between them is too short to
1 0 form a single-chain variable region fragment. As a result, the polypeptide chains form a dimer,
and thus the diabody has two antigen binding sites.
[0023]
sc(Fv) 2 is a single-chain minibody produced by linking two units of VH and two units of
VL with linkers and such (Hudson et aL, J Immunol. Methods (1999) 231 : 177-1 89). sc(Fv) 2
1 5 exhibits a particularly high agonistic activity compared to the whole antibody and other
minibodies. sc(Fv)2 can be produced, for example, by linking two scFv molecules.
In a preferable antibody, the two VH units and two VL units are arranged in the order of
VH, VL, VH, and VL ([VH]4inker-[VL]-linker-[VH]-linker-[VL]) beginning from the N
terminus of a single-chain polypeptide.
20 [0024]
The order of the two VH units and two VL units is not limited to the above arrangement,
and they may be arranged in any order. Examples of the arrangements are listed below.
|VL]-linker-|yH]-linker-|yH]-linker-|yL]
|yH]-linker-|yL]-linker-|yL]-linker-|yH]
25 |yH]-linker-|yH]-linker-[VL]-linker-[VL]
|yL]-linker-[VL]-linker-[VH]-linker-[VH]
|yL]-linker-|yH]-linker-|yL]-linker-|yH]
[0025]
The linkers to be used for linking the variable regions of an antibody comprise arbitrary
30 peptide linkers that can be introduced by genetic engineering, synthetic linkers, and linkers
disclosed in, for example, Holliger, P. et aL, Protein Engineering (1996) 9 (3): 299-305.
Peptide linkers are preferred in the present invention. There are no limitations as to the length
of the peptide linkers. The length can be selected accordingly by those skilled in the art
depending on the purpose, and is typically 1-100 amino acids, preferably 3-50 amino acids, more
35 preferably 5-30 amino acids, and even more preferably 12-18 amino acids (for example, 15
amino acids).
21 JP 2004-071763
[0026]
For example, such peptide linkers include:
Ser
Gly Ser
5 Gly Gly Ser
Ser Gly Gly
Gly Gly Gly Ser
Ser Gly Gly Gly
Gly Gly Gly Gly Ser
10 Ser Gly Gly Gly Gly
Gly Gly Gly Gly Gly Ser
Ser Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Ser
Ser Gly Gly Gly Gly Gly Gly
1 5 (Gly Gly Gly Gly Ser) n
(Ser Gly Gly Gly Gly) n
where n is an integer of 1 or larger. The lengths and sequences of peptide linkers can
be selected accordingly by those skilled in the art depending on the purpose.
In an embodiment of the present invention, a particularly preferable sc(Fv)2 includes,
20 for example, the sc(Fv)2 below.
[0027]
[VH] -peptide linker (15 amino acids)- [VL] -peptide linker (15 amino
acids)-[VH]-peptide linker (15 amino acids)-[VL]
[0028]
25 Synthetic linkers (chemical crosslinking agents) include crosslinking agents routinely
used to crosslink peptides, for example, N-hydroxy succinimide (NHS), disuccinimidyl suberate
(DSS), bis(sulfosuccinimidyl) suberate (BS 3 ), dithiobis(succinimidyl propionate) (DSP),
dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate)
(EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate
30 (DST), dismfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]
sulfone (BSOCOES), and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone
(sulfo-BSOCOES). These crosslinking agents are commercially available.
[0029]
In general, three linkers are required to link four antibody variable regions together.
35 The linkers to be used may be of the same type or different types. In the present invention, a
preferable minibody is a diabody, even more preferably, an sc(Fv) 2 . Such a minibody can be
22
JP 2004-071763
prepared by treating an antibody with an enzyme, for example, papain or pepsin, to generate
antibody fragments, or by constructing DNAs encoding those antibody fragments and
introducing them into expression vectors, followed by expression in an appropriate host cell (see,
for example, Co, M. S. etal.J. Immunol. (1994) 152: 2968-2976; Better, M and Horwitz, A. H.,
5 Methods Enzymol. (1989) 178: 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol.
(1989) 178: 497-515; Lamoyi, E., Methods Enzymol. (1986) 121: 652-663; Rousseaux, J. etal,
Methods Enzymol. (1986) 121 : 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol.
(1991)9: 132-137).
[0030]
10 An antibody having exceedingly high agonistic activity can be prepared by reducing the
molecular weight of a full-length antibody, particularly by converting it into an sc(Fv)2.
In a preferred embodiment, the antibodies of the present invention comprise modified
antibodies, such as chimeric antibodies and humanized antibodies that bind to Mpl. These
modified antibodies can be produced by known methods.
15 [0031]
Chimeric antibodies are antibodies prepared by combining sequences derived from
different animal species, and include for example, antibodies comprising the heavy chain and
light chain variable regions of a murine antibody, and the heavy chain and light chain constant
regions of a human antibody. Chimeric antibodies can be prepared by known methods. For
20 example, a DNA encoding the V region of an antibody is linked to a DNA encoding the C region
of a human antibody, and the construct is inserted into an expression vector and introduced into a
host to produce chimeric antibodies.
[0032]
Humanized antibodies are also referred to as "reshaped human antibodies". Such a
25 humanized antibody is obtained by transferring the complementarity-determining region (CDR)
of an antibody derived from a non-human mammal, for example mouse, to the
complementarity-determining region of a human antibody, and the general gene recombination
procedure for this is also known (see European Patent Application No. 125023 and WO
96/02576).
30 [0033]
Specifically, a DNA sequence designed to link a murine antibody CDR to the
framework region (FR) of a human antibody can be synthesized by PCR, using primers prepared
from several oligonucleotides containing overlapping portions of both CDR and FR terminal
regions (see methods described in WO 98/13388).
35 [0034]
The human antibody framework region to be linked by CDR is selected in order to form
23 JP 2004-071763
a favorable antigen-binding site in the complementarity-determining region. Amino acids of
the framework region in the antibody variable region may be substituted, as necessary, for the
complementarity-determining region of the reshaped human antibody to form a suitable
antigen-binding site (Sato, K. et al. 9 Cancer Res. (1993) 53: 851-856).
5 [0035]
The constant region of a human antibody is used as the constant region of a chimeric
antibody or humanized antibody. For example, Cyl, Cy2, Cy3, and Cy4 can be used as the H
chain, and Ck and CA, can be used as the L chain. The human antibody constant region may be
modified to improve the antibody or the stability of the antibody production.
10 [0036]
Generally, chimeric antibodies comprise the variable region of an antibody from a
non-human mammal and the constant region derived from a human antibody. On the other
hand, humanized antibodies comprise the complementarity-determining region of an antibody
from a non-human mammal, and the framework region and constant region derived from a
1 5 human antibody.
[0037]
In addition, after a chimeric antibody or a humanized antibody is prepared, amino acids
in the variable region (for example, FR) and the constant region may be replaced with other
amino acids, and such.
20 The origin of the variable regions in chimeric antibodies or that of the CDRs in
humanized antibodies is not particularly limited, and may be derived from any type of animal.
For example, sequences of murine antibodies, rat antibodies, rabbit antibodies, camel antibodies
may be used.
[0038]
25 In general, it is difficult to chimerize or humanize an antibody without losing the
agonistic activity of the original antibody. Nevertheless, the present invention succeeded in
preparing humanized antibodies having agonistic activity equivalent to that of the original
murine antibody.
[0039]
30 A preferred humanized antibody of the present invention is an antibody comprising a
heavy chain variable region that comprises the amino acid sequence of SEQ ID NO: 229
(humanized heavy chain sequence: hVB22B p-z VH), SEQ ID NO: 256 (humanized heavy chain
sequence: hVB22B g-e VH), or SEQ ID NO: 262 (humanized heavy chain sequence: hVB22B e
VH); or an antibody comprising a light chain variable region that comprises the amino acid
35 sequence of SEQ ID NO: 238 (humanized light chain hVB22B p-z VL), or SEQ ID NO: 258
(humanized light chain hVB22B g-e VL or hVB22B e VL). In particular, a preferred antibody
24
JP 2004-071763
is an antibody comprising a heavy chain variable region and a light chain variable region of any
one of (1) to (3) indicated below:
(1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 229, and a
light chain variable region comprising the amino acid sequence of SEQ ID NO: 238;
5 (2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 256, and a
light chain variable region comprising the amino acid sequence of SEQ ID NO: 258;
(3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 262, and a
light chain variable region comprising the amino acid sequence of SEQ ID NO: 258.
[0040]
10 Such antibodies include, for example, antibodies comprising the amino acid sequence of
SEQ ID NO: 2, 254, or 260 (humanized sc(Fv) 2 sequence (hVB22B p-z sc(Fv) 2 , hVB22B g-e
sc(Fv) 2 , or hVB22B e sc(Fv) 2 ).
[0041]
The nucleotide sequence of hVB22B p-z VH is shown in SEQ ID NO: 228; the
15 nucleotide sequence of hVB22B g-e VH is shown in SEQ ID NO: 255; the nucleotide sequence
of hVB22B e VH is shown in SEQ ID NO: 261 ; the nucleotide sequence of hVB22B p-z VL is
shown in SEQ ID NO: 237; the nucleotide sequences of hVB22B g-e VL and hVB22B e VL are
shown in SEQ ID NO: 257.
[0042]
20 In the amino acid sequence of SEQ ID NO: 229 (humanized heavy chain sequence:
hVB22B p-z VH), SEQ ID NO: 256 (humanized heavy chain sequence: hVB22B g-e VH), or
SEQ ID NO: 262 (humanized heavy chain sequence: hVB22B e VH),
amino acids 31-35 correspond to CDR1;
amino acids 50-66 correspond to CDR2;
25 amino acids 99-107 correspond to CDR3;
amino acids 1-30 correspond to FR1;
amino acids 36-49 correspond to FR2;
amino acids 67-98 correspond to FR3; and
amino acids 108-118 correspond to FR4.
30 [0043]
In the amino acid sequence of SEQ ID NO: 238 (humanized light chain sequence:
hVB22B p-z VL) or SEQ ID NO: 258 (humanized light chain sequence: hVB22B g-e VL or
hVB22B e VL),
amino acids 24-39 correspond to CDR1;
35 amino acids 55-61 correspond to CDR2;
amino acids 94-102 correspond to CDR3;
25
JP 2004-071763
amino acids 1-23 correspond to FR1;
amino acids 40-54 correspond to FR2;
amino acids 62-93 correspond to FR3 ; and
amino acids 103-112 correspond to FR4.
5 [0044]
In the present invention, SEQ ID NOs of the CDRs and FRs in the hVB22B p-z VH
sequence are shown below:
hVB22B p-z VH: FR1/SEQ ID NO: 230
hVB22B p-z VH: CDR1/SEQ ID NO: 23 1
1 0 hVB22B p-z VH: FR2/SEQ ID NO: 232
hVB22B p-z VH: CDR2/SEQ ID NO : 23 3
hVB22B p-z VH: FR3/SEQ ID NO: 234
hVB22B p-z VH: CDR3/SEQ ID NO: 235
hVB22B p-z VH: FR4/SEQ ID NO: 236.
15 [0045]
In the present invention, SEQ ID NOs of the CDRs and FRs in the hVB22B p-z VL
sequence are shown below:
hVB22B p-z VL: FR1/SEQ ID NO: 239
hVB22B p-z VL: CDR1/SEQ ID NO: 240
20 hVB22B p-z VL: FR2/SEQ ID NO: 241
hVB22B p-z VL: CDR2/SEQ ID NO: 242
hVB22B p-z VL: FR3/SEQ ID NO: 243
hVB22B p-z VL: CDR3/SEQ ID NO: 244
hVB22B p-z VL: FR4/SEQ ID NO: 245.
25 [0046]
In the present invention, SEQ ID NOs of the CDRs and FRs in the hVB22B g-e VH
sequence are shown below:
hVB22B g-e VH: FR1/SEQ ID NO: 265
hVB22B g-e VH: CDR1/SEQ ID NO: 266
30 hVB22B g-e VH: FR2/SEQ ID NO: 267
hVB22B g-e VH: CDR2/SEQ ID NO: 268
hVB22B g-e VH: FR3/SEQ ID NO: 269
hVB22B g-e VH: CDR3/SEQ ID NO: 270
hVB22B g-e VH: FR4/SEQ ID NO: 271.
35 [0047]
In the present invention, SEQ ID NOs of the CDRs and FRs in the hVB22B g-e VL
26
JP 2004-071763
sequence are shown below:
hVB22B g-e VL: FR1/SEQ ID NO: 272
hVB22B g-e VL: CDR1/SEQ ID NO: 273
hVB22B g-e VL: FR2/SEQ ID NO: 274
5 hVB22B g-e VL: CDR2/SEQ ID NO: 275
hVB22B g-e VL: FR3/SEQ ID NO: 276
hVB22B g-e VL: CDR3/SEQ ID NO: 277
hVB22B g-e VL: FR4/SEQ ID NO: 278.
[0048]
10 In the present invention, SEQ ID NOs of the CDRs and FRs in the hVB22B e VH
sequence are shown below:
hVB22B e VH: FR1/SEQ ID NO: 279
hVB22B e VH: CDR1/SEQ ID NO: 280
hVB22B e VH: FR2/SEQ ID NO: 281
1 5 hVB22B e VH: CDR2/SEQ ID NO: 282
hVB22B e VH: FR3/SEQ ID NO: 283
hVB22B e VH: CDR3/SEQ ID NO: 284
hVB22B e VH: FR4/SEQ ID NO: 285.
[0049]
20 In the present invention, SEQ ID NOs of the CDRs and FRs in the hVB22B e VL
sequence are shown below:
hVB22B e VL: FR1/SEQ ID NO: 272
hVB22B e VL: CDR1/SEQ ID NO: 273
hVB22B e VL: FR2/SEQ ID NO: 274
25 hVB22B e VL: CDR2/SEQ ID NO: 275
hVB22B e VL: FR3/SEQ ID NO: 276
hVB22B e VL: CDR3/SEQ ID NO: 277
hVB22B e VL: FR4/SEQ ID NO: 278.
[0050]
30 SEQ ID NOs of the CDRs and FRs in the hVB22Bp-zVH sequence and hVB22B p-z
VL sequence are shown in Figure 18.
[0051]
In other embodiments, preferred humanized antibodies of the present invention include:
humanized antibodies comprising a heavy chain variable region which has FR1, 2, 3, and 4
35 comprising amino acid sequences of any one of (1) to (3) indicated below:
(1) SEQ ED NOs: 230, 232, 234, and 236,
27
JP 2004-071763
(2) SEQ ID NOs: 265, 267, 269, and 271,
(3) SEQ ID NOs: 279, 281, 283, and 285;
humanized antibodies comprising a light chain variable region which has FR1, 2, 3, and 4
comprising amino acid sequences of (1) or (2) listed below:
5 (1) SEQ ID NOs: 239, 241, 243, and 245,
(2) SEQ ID NOs: 272, 274, 276, and 278;
humanized antibodies comprising a heavy chain variable region which has CDR1, 2 and 3
comprising amino acid sequences of any one of (1) to (3) listed below:
(1) SEQ ID NOs: 231, 233, and 235,
10 (2) SEQ ID NOs: 266, 268, and 270,
(3) SEQ ID NOs: 280, 282, and 284; and
humanized antibodies comprising a light chain variable region which has CDR1 , 2 and 3
comprising amino acid sequences of (1) or (2) listed below:
(1) SEQ ID NOs: 240, 242, and 244,
15 (2) SEQ ID NOs: 273, 275, and 277.
[0052]
In yet another preferred embodiments, preferred humanized antibodies of the present
invention include:
humanized antibodies comprising heavy chain and light chain variable regions of any one of (1)
20 to (3) indicated below.
(1) a heavy chain variable region which comprises FR1, 2, 3, and 4 comprising the amino acid
sequences of SEQ ID NOs: 230, 232, 234, and 236, respectively, and a light chain variable
region which comprises FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
239, 241 , 243, and 245, respectively;
25 (2) a heavy chain variable region which comprises FR1, 2, 3, and 4 comprising the amino acid
sequences of SEQ ID NOs: 265, 267, 269, and 271, respectively, and a light chain variable
region which comprises FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
272, 274, 276, and 278, respectively;
(3) a heavy chain variable region which comprises FR1, 2, 3 and 4 comprising the amino acid
30 sequences of SEQ ID NOs: 279, 281, 283, and 285, respectively, and a light chain variable
region which comprises FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs:
272, 274, 276, and 278, respectively; and
humanized antibodies comprising heavy chain and light chain variable regions of any one of (1)
to (3) indicated below:
35 (1) a heavy chain variable region which comprises CDR1, 2, and 3 comprising the amino acid
sequences of SEQ ID NOs: 231, 233, and 235, respectively, and a light chain variable region
28 JP 2004-071763
which comprises CDR1, 2, and 3 comprising the amino acid sequences of SEQ ID NOs: 240,
242, and 244, respectively;
(2) a heavy chain variable region which comprises CDR1, 2, and 3 comprising the amino acid
sequences of SEQ ID NOs: 266, 268, and 270, respectively, and a light chain variable region
5 which comprises CDR1, 2, and 3 comprising the amino acid sequences of SEQ ID NOs: 273,
275, and 277, respectively;
(3) a heavy chain variable region which comprises CDR1, 2, and 3 comprising the amino acid
sequences of SEQ ID NOs: 280, 282, and 284, respectively, and a light chain variable region
which comprises CDR1, 2, and 3 comprising the amino acid sequences of SEQ ID NOs: 273,
10 275, and 277, respectively.
[0053]
Chimeric antibodies and humanized antibodies exhibit lower antigenicity in the human
body, and thus are expected to be useful when administered to humans for therapeutic purposes.
In one embodiment, the preferred antibodies of the present invention include antibodies
15 that bind to soluble Mpl. The term "soluble Mpl" herein refers to Mpl molecules excluding
those expressed on the cell membrane. A specific example of a soluble Mpl is an Mpl lacking
the entire or a portion of the transmembrane domain. The transmembrane domain of human
Mpl corresponds to amino acids 492 to 513 in SEQ ID NO: 123.
[0054]
20 An antibody that binds to soluble recombinant Mpl can be used in detailed epitope
analysis and kinetic analysis of receptor-ligand binding, as well as for assessing the blood
concentration and dynamic behavior of the antibody in in vivo tests.
In one embodiment, the preferred antibodies of the present invention include antibodies
having binding activity against both human and monkey Mpl. Antibodies having agonistic
25 activity to both human and monkey Mpl are expected to be highly useful since the dynamic
behavior and in vivo effects of the antibody, which are generally difficult to determine in human
body, can be examined with monkeys.
[0055]
The present invention also provides antibodies having agonistic activity to human Mpl
30 and monkey Mpl. Such antibodies may also have binding activity or agonistic activity against
Mpl from animals other than humans and monkeys (for example, mice).
[0056]
In addition, the antibodies of the present invention include antibodies with TPO
agonistic activity (agonistic activity against Mpl) of EC50 = 100 nM or lower, preferably EC50 =
35 30 nM or lower, more preferably EC50 = 10 nM or lower.
[0057]
29 JP 2004-071763
The agonistic activity can be determined by methods known to those skilled in the art,
for example, by the method described below.
The sequences for human Mpl (Palacios et aL, Cell (1985) 41 : 727-734; GenBank Accession NO.
NM_005373), cynomolgus monkey Mpl (SEQ ID NO: 157), and mouse Mpl (GenBank
5 Accession NO. NM_010823) are already known.
[0058]
In addition, the present invention includes antibodies whose binding activities to soluble
Mpl are KD = 1 0" 6 M or lower, preferably KD = 1 0" 7 M or lower, and more preferably KD = 1 0" 8
M or lower.
10 [0059]
In the present invention, whether the binding activity of an antibody to soluble
recombinant Mpl is KD = 1 0" 6 M or lower can be determined by methods known to those skilled
in the art. For example, the activity can be determined using surface plasmon resonance with
Biacore. Specifically, soluble MPL-Fc protein is immobilized onto sensor chips. Reaction
1 5 rate constant can be determined by assessing the interaction between the antibody and the
soluble Mpl-Fc protein. The binding activity can be evaluated by ELISA (enzyme -linked
immunosorbent assays), EIA (enzyme immunoassays), RIA (radio immunoassays), or
fluorescent antibody techniques. For example, in enzyme immunoassays, a sample containing a
test antibody, such as purified antibody or culture supernatant of cells producing the test antibody,
20 is added to a plate coated with an antigen to which the test antibody can bind. After incubating
the plate with a secondary antibody labeled with an enzyme such as alkaline phosphatase, the
plate is washed and an enzyme substrate such as p-nitrophenyl phosphate is added. The
antigen-binding activity can then be evaluated by determining the absorbance.
[0060]
25 There is no specific limitation as to the upper limit of the binding activity; for example,
the upper limit may be set within a technically feasible range by those skilled in the art.
However, the technically feasible range may expand with the advancement of technology.
[0061]
In an embodiment, the preferred antibodies of the present invention include antibodies
30 recognizing epitopes that are recognized by any one of the antibodies indicated in (I) to (XII)
below. The antibody of any one of (I) to (XII) is preferably a minibody.
[0062]
(I)
Antibody comprising a VH that has CDR1, 2, and 3 comprising the amino acid
35 sequences according to SEQ ID NOs in any one of (1) to (22) indicated below (name of each
antibody and the H chain CDR contained in the antibody are indicated inside the parentheses):
30
JP 2004-071763
(1) SEQ ID NOs: 3, 4, and 5 (VA7: H chain CDR1, 2, and 3),
(2) SEQ ID NOs: 6, 7, and 8 (VA130: H chain CDR1, 2, and 3),
(3) SEQ ID NOs: 9, 10, and 11 (VA259: H chain CDR1, 2, and 3),
(4) SEQ ID NOs: 12, 13, and 14 (VB17B: H chain CDR1, 2, and 3),
5 (5) SEQ ID NOs: 15, 16, and 17 (VB12B: H chain CDR1, 2, and 3),
(6) SEQ ID NOs: 18, 19, and 20 (VB140: H chain CDR1, 2, and 3),
(7) SEQ ID NOs: 21, 22, and 23 (VB33: H chain CDR1, 2, and 3),
(8) SEQ ID NOs: 24, 25, and 26 (VB45B: H chain CDR1 , 2, and 3),
(9) SEQ ID NOs: 27, 28, and 29 (VB8B: H chain CDR1, 2, and 3),
10 (10) SEQ ID NOs: 30, 31, and 32 (VB115: H chain CDR1, 2, and 3),
(11) SEQ ID NOs: 33, 34, and 35 (VB14B: H chain CDR1, 2, and 3),
(12) SEQ ID NOs: 36, 37, and 38 (VB22B: H chain CDR1, 2, and 3),
(13) SEQ ID NOs: 39, 40, and 41 (VB16: H chain CDR1, 2, and 3),
(14) SEQ ID NOs: 42, 43, and 44 (VB157: H chain CDR1, 2, and 3),
15 (15) SEQ ID NOs: 45, 46, and 47 (VB4B: H chain CDR1, 2, and 3),
(16) SEQ ID NOs: 48, 49, and 50 (VB51: H chain CDR1, 2, and 3),
(17) SEQ ID NOs: 51, 52, and 53 (AB317: H chain CDR1, 2, and 3),
(18) SEQ ID NOs: 54, 55, and 56 (AB324: H chain CDR1, 2, and 3),
(19) SEQ ID NOs: 57, 58, and 59 (TA136: H chain CDR1, 2, and 3),
20 (20) SEQ ID NOs: 23 1 , 233, and 235 (hVB22B p-z: H chain CDR1 , 2, and 3),
(21) SEQ ID NOs: 266, 268, and 270 (hVB22B g-e: H chain CDR1, 2, and 3),
(22) SEQ ID NOs: 280, 282, and 284 (hVB22B e: H chain CDR1, 2, and 3).
[0063]
(II)
25 Antibody comprising a VL which has CDR1 , 2, and 3 comprising the amino acid
sequences according to SEQ ID NOs in any one of (1) to (21) indicated below (name of each
antibody and the L chain CDR in the antibody are indicated inside the parentheses):
(1) SEQ ID NOs: 60, 61, and 62 (VA7: L chain CDR1, 2, and 3),
(2) SEQ ID NOs: 63, 64, and 65 (VA130: L chain CDR1, 2, and 3),
30 (3) SEQ ID NOs: 66, 67, and 68 (VA259: L chain CDR1, 2, and 3),
(4) SEQ ID NOs: 69, 70, and 71 (VB17B: L chain CDR1, 2, and 3),
(5) SEQ ID NOs: 72, 73, and 74 (VB12B: L chain CDR1, 2, and 3),
(6) SEQ ID NOs: 75, 76, and 77 (VB140: L chain CDR1, 2, and 3)
(7) SEQ ID NOs: 78, 79, and 80 (VB33: L chain CDR1, 2, and 3),
35 (8) SEQ ID NOs: 81, 82, and 83 (VB45B: L chain CDR1, 2, and 3),
(9) SEQ ID NOs: 84, 85, and 86 (VB8B: L chain CDR1, 2, and 3),
31
JP 2004-071763
(10) SEQ ID NOs: 87, 88, and 89 (VB115: L chain CDR1, 2, and 3),
(11) SEQ ID NOs: 90, 91, and 92 (VB14B: L chain CDR1, 2, and 3),
(12) SEQ ID NOs: 93, 94, and 95 (VB22B: L chain CDR1, 2, and 3),
(13) SEQ ID NOs: 96, 97, and 98 (VB16: L chain CDR1, 2, and 3),
5 (14) SEQ ID NOs: 99, 100, and 101 (VB157: L chain CDR1, 2, and 3),
(15) SEQ ID NOs: 102, 103, and 104 (VB4B: L chain CDR1, 2, and 3),
(16) SEQ ID NOs: 105, 106, and 107 (VB51: L chain CDR1, 2, and 3),
(17) SEQ ID NOs: 108, 109, and 110 (AB317: L chain CDR1, 2, and 3),
(18) SEQ ID NOs: 111, 112, and 113 (AB324: L chain CDR1, 2, and 3),
10 (19) SEQ ID NOs: 114, 115, and 116 (TA136: L chain CDR1, 2, and 3),
(20) SEQ ID NOs: 240, 242, and 244 (hVB22B p-z: L chain CDR1, 2, and 3),
(21) SEQ ID NOs: 273, 275, and 277 (hVB22B g-e or hVB22B e: L chain CDR1, 2, and 3).
[0064]
(III)
1 5 Antibody comprising a VH that comprises an amino acid sequence of the SEQ ID NO in
any one of (1) to (22):
(1) SEQ ID NO: 124 (VA7: VH),
(2) SEQ ID NO: 126 (VA130: VH),
(3) SEQ ID NO: 128 (VA259: VH),
20 (4) SEQ ID NO: 130 (VB17B: VH),
(5) SEQ ID NO: 132 (VB12B: VH),
(6) SEQ ID NO: 134 (VB140: VH),
(7) SEQ ID NO: 136 (VB33: VH),
(8) SEQ ID NO: 138 (VB45B: VH),
25 (9) SEQ ID NO: 140 (VB8B: VH),
(10) SEQ ID NO: 142 (VB115:VH),
(11) SEQ ID NO: 144 (VB14B: VH),
(12) SEQ ID NO: 118 (VB22B: VH),
(13) SEQ ID NO: 146 (VB16: VH),
30 (14) SEQ ID NO: 148 (VB157: VH),
(15) SEQ ID NO: 150 (VB4B: VH),
(16) SEQ ID NO: 152 (VB51: VH),
(17) SEQ ID NO: 155 (AB317: VH),
(18) SEQ ID NO: 159 (AB324: VH),
35 (19) SEQ ID NO: 162 (TA136: VH),
(20) SEQ ID NO: 229 (hVB22B p-z: VH),
32 JP 2004-071763
(21) SEQ ID NO: 256 (hVB22B g-e: VH),
(22) SEQ ID NO: 262 (hVB22B e: VH).
[0065]
(IV)
5 Antibody comprising a VL that comprises an amino acid sequence of the SEQ ID NO in
any one of (1) to (21):
(1) SEQ ID NO: 125 (VA7: VL),
(2) SEQ ID NO: 127 (VA130: VL),
(3) SEQ ID NO: 129 (VA259: VL),
10 (4) SEQ ID NO: 131 (VB17B: VL),
(5) SEQ ID NO: 133 (VB12B: VL),
(6) SEQ ID NO: 135 (VB140: VL),
(7) SEQ ID NO: 137 (VB33: VL),
(8) SEQ ID NO: 139 (VB45B: VL),
15 (9) SEQ ID NO: 141 (VB8B: VL),
(10) SEQ ID NO: 143 (VB115: VL),
(11) SEQ ID NO: 145 (VB14B: VL),
(12) SEQ ID NO: 120 (VB22B: VL),
(13) SEQ ID NO: 147 (VB16: VL),
20 (14) SEQ ID NO: 149 (VB157: VL),
(15) SEQ ID NO: 151 (VB4B: VL),
(16) SEQ ID NO: 153 (VB51: VL),
(17) SEQ ID NO: 157 (AB317: VL),
(18) SEQ ID NO: 161 (AB324: VL),
25 (19) SEQ ID NO: 163 (TA136: VL),
(20) SEQ ID NO: 238 (hVB22B p-z: VL),
(21) SEQ ID NO: 258 (hVB22B g-e: VL or hVB22B e: VL).
[0066]
(V)
30 Antibody comprising a VH and VL according to any one of (1) to (22):
(1) SEQ ID NOs: 3, 4, and 5 (VA7: H chain CDR1, 2, and 3); SEQ ID NOs: 60, 61, and 62
(VA7: L chain CDR1, 2, and 3),
(2) SEQ ID NOs: 6, 7, and 8 (VA130: H chain CDR1, 2, and 3), SEQ ID NOs: 63, 64, and 65
(VA130: L chain CDR1, 2, and 3),
35 (3) SEQ ID NOs: 9, 10, and 1 1 (VA259: H chain CDR1, 2, and 3); SEQ ED NOs: 66, 67, and 68
(VA259: L chain CDR1, 2, and 3),
33
JP 2004-071763
(4) SEQ ID NOs: 12, 13, and 14 (VB17B: H chain CDR1, 2, and 3); SEQ ID NOs: 69, 70, and
71 (VB17B: L chain CDR1, 2, and 3),
(5) SEQ ID NOs: 15, 16, and 17 (VB12B: H chain CDR1, 2, and 3); SEQ ID NOs: 72, 73, and
74 (VB12B: L chain CDR1, 2, and 3),
5 (6) SEQ ID NOs: 18, 19, and 20 (VB140: H chain CDR1, 2, and 3); SEQ ID NOs: 75, 76, and 77
(VB140: L chain CDR1, 2, and 3),
(7) SEQ ID NOs: 21, 22, and 23 (VB33: H chain CDR1, 2, and 3); SEQ ID NOs: 78, 79, and 80
(VB33: L chain CDR1, 2, and 3),
(8) SEQ ID NOs: 24, 25, and 26 (VB45B: H chain CDR1, 2, and 3); SEQ ID NOs: 81, 82, and
10 83 (VB45B: L chain CDR1, 2, and 3),
(9) SEQ ID NOs: 27, 28, and 29 (VB8B: H chain CDR1, 2, and 3); SEQ ID NOs: 84, 85, and 86
(VB8B: L chain CDR1, 2, and 3),
(10) SEQ ID NOs: 30, 31, and 32 (VB115: H chain CDR1, 2, and 3); SEQ ID NOs: 87, 88, and
89 (VB115: L chain CDR1, 2, and 3),
15 (11) SEQ ID NOs: 33, 34, and 35 (VB14B: H chain CDR1, 2, and 3); SEQ ID NOs: 90, 91, and
92 (VB14B: L chain CDR1, 2, and 3),
(12) SEQ ID NOs: 36, 37, and 38 (VB22B: H chain CDR1, 2, and 3); SEQ ID NOs: 93, 94, and
95 (VB22B: L chain CDR1, 2, and 3),
(13) SEQ ID NOs: 39, 40, and 41 (VB16: H chain CDR1, 2, and 3); SEQ ID NOs: 96, 97, and 98
20 (VB 1 6: L chain CDR1 , 2, and 3),
(14) SEQ ID NOs: 42, 43, and 44 (VB157: H chain CDR1, 2, and 3); SEQ ID NOs: 99, 100, and
101 (VB157: L chain CDR1, 2, and 3),
(15) SEQ ID NOs: 45, 46, and 47 (VB4B: H chain CDR1, 2, and 3); SEQ ID NOs: 102, 103, and
104 (VB4B: L chain CDR1, 2, and 3),
25 (16) SEQ ID NOs: 48, 49, and 50 (VB51: H chain CDR1, 2, and 3); SEQ ID NOs: 105, 106, and
107 (VB51 : L chain CDR1, 2, and 3),
(17) SEQ ID NOs: 51, 52, and 53 (AB317: H chain CDR1, 2, and 3); SEQ ID NOs: 108, 109,
and 110 (AB317: L chain CDR1, 2, and 3),
(18) SEQ ID NOs: 54, 55, and 56 (AB324: H chain CDR1, 2, and 3); SEQ ID NOs: 111,112,
30 and 1 1 3 (AB324: L chain CDR1 , 2, and 3),
(19) SEQ ID NOs: 57, 58, and 59 (TA136: H chain CDR1, 2, and 3); SEQ ID NOs: 114, 115, and
116 (TA136: L chain CDR1, 2, and 3),
(20) SEQ ID NOs: 231, 233, and 235 (hVB22B p-z: H chain CDR1, 2, and 3); SEQ ID NOs: 240,
242, and 244 (hVB22B p-z: L chain CDR1, 2, and 3),
35 (21) SEQ ID NOs: 266, 268, and 270 (hVB22B g-e: H chain CDR1, 2, and 3); SEQ ID NOs: 273,
275, and 277 (hVB22B g-e: L chain CDR1, 2, and 3),
34
JP 2004-071763
(22) SEQ ID NOs: 280, 282, and 284 (hVB22B e: H chain CDR1, 2, and 3); SEQ ID NOs: 273,
275, and 277 (hVB22B e: L chain CDR1, 2, and 3).
[0067]
(VI)
5 Antibody comprising a VH and a VL that comprise the amino acid sequences according
to SEQ ID NOs in any one of (1) to (22) indicated below:
(1) SEQ ID NO: 124 (VA7: VH), SEQ ID NO: 125 (VA7: VL),
(2) SEQ ID NO: 126 (VA130: VH), SEQ ID NO: 127 (VA130: VL),
(3) SEQ ID NO: 128 (VA259: VH), SEQ ID NO: 129 (VA259: VL),
10 (4) SEQ ID NO: 130 (VB17B: VH), SEQ ID NO: 131 (VB17B: VL),
(5) SEQ ID NO: 132 (VB12B: VH), SEQ ID NO: 133 (VB12B: VL),
(6) SEQ ID NO: 134 (VB140: VH), SEQ ID NO: 135 (VB140: VL),
(7) SEQ ID NO: 136 (VB33: VH), SEQ ID NO: 137 (VB33: VL),
(8) SEQ ID NO: 138 (VB45B: VH), SEQ ID NO: 139 (VB45B: VL),
15 (9) SEQ ID NO: 140 (VB8B: VH), SEQ ID NO: 141 (VB8B: VL),
(10) SEQ ID NO: 142 (VB115: VH), SEQ ID NO: 143 (VB115:VL),
(11) SEQ ID NO: 144 (VB14B: VH), SEQ ID NO: 145 (VB14B: VL),
(12) SEQ ID NO: 118 (VB22B: VH), SEQ ID NO: 120 (VB22B: VL),
(13) SEQ ID NO: 146 (VB16: VH), SEQ ID NO: 147 (VB16: VL),
20 (14) SEQ ID NO: 148 (VB157: VH), SEQ ID NO: 149 (VB157: VL),
(15) SEQ ID NO: 150 (VB4B: VH), SEQ ID NO: 151 (VB4B: VL),
(16) SEQ ID NO: 152 (VB51: VH), SEQ ID NO: 153 (VB51: VL),
(17) SEQ ID NO: 155 (AB317: VH), SEQ ID NO: 157 (AB317: VL),
(18) SEQ ID NO: 159 (AB324: VH), SEQ ID NO: 161 (AB324: VL),
25 (19) SEQ ID NO: 162 (TA136: VH), SEQ ID NO: 163 (TA136: VL),
(20) SEQ ID NO: 229 (hVB22B p-z: VH), SEQ ID NO: 238 (hVB22B p-z: VL),
(21) SEQ ID NO: 256 (hVB22B g-e: VH), SEQ ID NO: 258 (hVB22B g-e: VL),
(22) SEQ ID NO: 262 (hVB22B e: VH), SEQ ID NO: 258 (hVB22B e: VL).
[0068]
30 (VII)
Antibody comprising the amino acid sequence of SEQ ID NO: 122 (VB22B: scFv).
[0069]
(VIII)
Humanized antibody comprising an amino acid sequence according to any one of SEQ
35 ID NO: 2 (hVB22B p-z: sc(Fv) 2 ), SEQ ID NO: 254 (hVB22B g-e: sc(Fv) 2 ), and SEQ ID NO:
260 (hVB22B e: sc(Fv) 2 ).
JP 2004-071763
[0070]
(IX)
Antibody comprising a VH which has FR1, 2, 3, and 4 comprising amino acid
sequences according to SEQ ID NOs in any one of (1) to (3) indicated below:
5 (1) SEQ ID NOs: 230, 232, 234, and 236 (hVB22B p-z: H chain FR1, 2, 3, and 4),
(2) SEQ ID NOs: 265, 267, 269, and 271 (hVB22B g-e: H chain FR1, 2, 3, and 4),
(3) SEQ ID NOs: 279, 281, 283, and 285 (hVB22B e: H chain FR1, 2, 3, and 4).
[0071]
(X)
1 0 Antibody comprising a VL which has FR1 , 2, 3 and 4 comprising amino acid sequences
according to SEQ ID NOs in any one of (1) or (2) indicated below:
(1) SEQ ID NOs: 239, 241, 243, and 245 (hVB22B p-z: L chain FR1, 2, 3, and 4),
(2) SEQ ID NOs: 272, 274, 276, and 278 (hVB22B g-e or hVB22B e: L chain FR1, 2, 3, and 4).
[0072]
15 (XI)
Antibody comprising VH and VL according to any one of (1) to (3) indicated below:
(1) VH having FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs: 230, 232,
234, and 236, respectively, and VL having FR1, 2, 3, and 4 comprising the amino acid sequences
of SEQ ID NOs: 239, 241, 243, and 245, respectively;
20 (2) VH having FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs: 265, 267,
269, and 271, respectively, and VL having FR1, 2, 3, and 4 comprising the amino acid sequences
of SEQ ID NOs: 272, 274, 276, and 278, respectively;
(3) VH having FR1, 2, 3, and 4 comprising the amino acid sequences of SEQ ID NOs: 279, 281,
283, and 285, respectively, and VL having FR1, 2, 3, and 4 comprising the amino acid sequences
25 of SEQ ID NOs: 272, 274, 276, and 278, respectively.
[0073]
(XII)
Antibody comprising the amino acid sequence of SEQ ID NO: 264 (VB22B: sc(Fv)2).
[0074]
30 An antibody comprising an amino acid sequence of any one of (I) to (XII) indicated
above, in which one or more amino acids have been substituted, deleted, added, and/or inserted,
wherein the antibody has activity equivalent to that of the antibody of any one of (I) to (XII).
[0075]
Herein, the phrase "functionally equivalent" means that an antibody of interest has a
35 biological or biochemical activity comparable to that of an antibody of the present invention.
Such activities include, for example, binding activities and agonistic activities.
36
JP 2004-071763
[0076]
Methods for preparing polypeptides functionally equivalent to a certain polypeptide are
well known to those skilled in the art, and include methods of introducing mutations into
polypeptides. For example, those skilled in the art can prepare an antibody functionally
5 equivalent to the antibodies of the present invention by introducing appropriate mutations into
the antibody using site-directed mutagenesis (Hashimoto-Gotoh, T. et al Gene (1995) 152:
271-275; Zoller, MJ, and Smith, M. Methods Enzymol. (1983) 100: 468-500; Kramer, W. et al,
Nucleic Acids Res. (1984) 12: 9441-9456; Kramer, W. and Fritz HJ, Methods Enzymol. (1987)
154: 350-367; Kunkel, TA, Proc. Natl. Acad. Sci. USA. (1985) 82: 488-492; Kunkel, Methods
10 Enzymol. (1988) 85: 2763-2766), or such. Amino acid mutations may occur naturally. Thus,
the present invention also comprises antibodies functionally equivalent to the antibodies of the
present invention and comprising the amino acid sequences of these antibodies, in which one or
more amino acids is mutated. Generally, the number of amino acids that are mutated is 50
amino acids or less, preferably 30 or less, more preferably 10 or less (for example, five amino
15 acids or less).
[0077]
An amino acid is preferably substituted for a different amino acid(s) that allows the
properties of the amino acid side-chain to be conserved. Examples of amino acid side chain
properties are: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V), hydrophilic amino acids
20 (R, D, N, C, E, Q, G, H, K, S, and T), amino acids comprising the following side chains: aliphatic
side chains (Q A, V, L, I, and P); hydroxyl -containing side chains (S, T, and Y);
sulfur-containing side chains (C and M); carboxylic acid- and amide-containing side chains (D,
N, E, and Q); basic side chains (R, K, and H); aromatic ring-containing side chains (H, F, Y, and
W) (amino acids are represented by one-letter codes in parentheses).
25 [0078]
A polypeptide comprising a modified amino acid sequence, in which one or more amino
acid residues is deleted, added, and/or replaced with other amino acids, is known to retain its
original biological activity (Mark, D. F. et al, Proc. Natl. Acad. Sci. USA (1984) 81: 5662-5666;
Zoller, M. J. & Smith, M. Nucleic Acids Research (1982) 10: 6487-6500; Wang, A. et al,
30 Science 224: 1431-1433; Dalbadie-McFarland, G etal, Proc. Natl. Acad. Sci. USA (1982) 79:
6409-6413).
[0079]
Fusion proteins containing antibodies that comprise the amino acid sequence of an
antibody of the present invention, in which two or more amino acid residues have been added,
35 are included in the present invention. The fusion protein results from a fusion between one of
the above antibodies and a second peptide or protein, and is included in the present invention.
37
JP 2004-071763
The fusion protein can be prepared by ligating a polynucleotide encoding an antibody of the
present invention and a polynucleotide encoding a second peptide or polypeptide in frame,
inserting this into an expression vector, and expressing the fusion construct in a host. Some
techniques known to those skilled in the art are available for this purpose. The partner peptide
5 or polypeptide to be fused with an antibody of the present invention may be a known peptide, for
example, FLAG (Hopp, T. P. et aL, BioTechnology (1988) 6: 1204-1210), 6x His consisting of
six His (histidine) residues, 1 Ox His, influenza hemagglutinin (HA), human c-myc fragment,
VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40 T antigen fragment, Ick tag,
a-tubulin fragment, B-tag, Protein C fragment. Other partner polypeptides to be fused with the
10 antibodies of the present invention include, for example, GST (glutathione-S-transferase), HA
(influenza hemagglutinin), immunoglobulin constant region, P-galactosidase, and MBP
(maltose-binding protein). A polynucleotide encoding one of these commercially available
peptides or polypeptides can be fused with a polynucleotide encoding an antibody of the present
invention. The fusion polypeptide can be prepared by expressing the fusion construct.
15 [0080]
As described below, the antibodies of the present invention may differ in amino acid
sequence, molecular weight, isoelectric point, presence/absence of sugar chains, and
conformation depending on the cell or host producing the antibody, or purification method.
However, a resulting antibody is included in the present invention, as long as it is functionally
20 equivalent to an antibody of the present invention. For example, when an antibody of the
present invention is expressed in prokaryotic cells, for example E. coli, a methionine residue is
added to the N terminus of the original antibody amino acid sequence. Such antibodies are
included in the present invention.
[0081]
25 An antibody that recognizes an epitope recognized by the antibody according to any one
of (I) to (XII) indicated above is expected to have a high agonistic activity. Such antibodies can
be prepared by methods known to those skilled in the art. The antibody can be prepared by, for
example, determining the epitope recognized by the antibody according to any one of (I) to (XII)
by conventional methods, and using a polypeptide comprising one of the epitope amino acid
30 sequences as an immunogen. Alternatively, the antibody can be prepared by determining the
epitopes of conventionally prepared antibodies and selecting an antibody that recognizes the
epitope recognized by an antibody of any one of (I) to (XII).
[0082]
In the present invention, a particularly preferred antibody is an antibody that recognizes
35 the epitope recognized by the antibody comprising the amino acid sequence of SEQ ID NO: 2.
The antibody comprising the amino acid sequence of SEQ ED NO: 2 is predicted to recognize the
38 JP 2004-071763
region from Glu 26 to Leu 274, preferably the region from Ala 189 to Gly 245, more preferably
the region from Gin 213 to Ala 23 1 of human Mpl. Thus, antibodies recognizing the region of
amino acids 26 to 274, or amino acids 189 to 245, or amino acids 213 to 23 1 of human Mpl are
also included in the present invention.
5 [0083]
Antibodies recognizing regions of amino acids 26 to 274, amino acids 189 to 245, or
amino acids 213 to 231 of the human Mpl amino acid sequence (SEQ ID NO: 123) can be
obtained by methods known to those skilled in the art. Such antibodies can be prepared by, for
example, using a peptide comprising amino acids 26 to 274, amino acids 189 to 245, or amino
10 acids 213 to 231 of the human Mpl amino acid sequence (SEQ ID NO: 123) as an immunogen.
Alternatively, such antibodies can be prepared by determining the epitope of a conventionally
prepared antibody and selecting an antibody that recognizes the same epitope recognized by an
antibody of the present invention.
[0084]
1 5 The present invention provides antibodies described above in (I) to (XII). In an
embodiment of the present invention, a preferred antibody is the one shown in (V), a more
preferred antibody is the one shown in (VI), and a still more preferred is the one shown in (VIII).
[0085]
The present invention also provides vectors comprising polynucleotides encoding the
20 antibodies of the present invention, or polynucleotides which hybridize under stringent
conditions to the polynucleotides of the present invention and encode antibodies having activities
equivalent to those of the antibodies of the present invention. The polynucleotides of the
present invention are polymers comprising multiple bases or base pairs of deoxyribonucleic
acids (DNA) or ribonucleic acids (RNA), and are not particularly limited, as long as they encode
25 the antibodies of the present invention. They may also contain non-natural nucleotides. The
polynucleotides of the present invention can be used to express antibodies using genetic
engineering techniques. The polynucleotides of this invention can also be used as probes in the
screening of antibodies functionally equivalent to the antibodies of the present invention.
Specifically, DNAs that hybridize under stringent conditions to a polynucleotide encoding an
30 antibody of the present invention, and encode antibodies having activity equivalent to those of
the antibodies of the present invention can be obtained by techniques such as hybridization and
gene amplification (for example, PCR), using a polynucleotide of the present invention or a
portion thereof as a probe. Such DNAs are also included in the polynucleotides of the present
invention. Hybridization techniques are well known to those skilled in the art (Sambrook, J et
35 al , Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1 989). Such
hybridization conditions include, for example, conditions of low stringency. Examples of
39 JP 2004-071763
conditions of low stringency include post-hybridization washing in 0.1 x SSC and 0.1% SDS at
42°C, and preferably in 0.1 x SSC and 0.1% SDS at 50°C. More preferable hybridization
conditions include those of high stringency. Highly stringent conditions include, for example,
washing in 5x SSC and 0.1% SDS at 65°C. In these conditions, the higher the temperature, the
5 higher the expectation of efficiently obtaining polynucleotides with a high homology. However,
several factors, such as temperature and salt concentration, can influence hybridization
stringency, and those skilled in the art can suitably select these factors to accomplish similar
stringencies.
[0086]
1 0 Antibodies that are encoded by polynucleotides obtained by the hybridization and gene
amplification techniques, and are functionally equivalent to the antibodies of the present
invention generally exhibit high homology to the antibodies of the this invention at the amino
acid level. The antibodies of the present invention include antibodies that are functionally
equivalent to the antibodies of the present invention, and exhibit high amino acid sequence
15 homology to the antibodies of this invention. The term "high homology" generally means
identity at the amino acid level of at least 50% or higher, preferably 75% or higher, more
preferably 85% or higher, still more preferably 95% or higher. Polypeptide homology can be
determined by the algorithm described in the report: Wilbur, W. J. and Lipman, D. J. Proc. Natl.
Acad. Sci. USA (1983) 80: 726-730.
20 [0087]
When E. coli is used as a host, there is no particular limitation as to the type of vector of
the present invention, as long as the vector contains an "ori" responsible for its replication in E.
coli and a marker gene. The "ori" ensures the amplification and mass production of the vector
in E. coli (for example, JM109, DH5a, HB101, and XL 1 Blue). The marker gene is used to
25 select the E. coli transformants (for example, a drug resistance gene selected by an appropriate
drug such as ampicillin, tetracycline, kanamycin, and chloramphenicol). The vectors include,
for example, Ml 3 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. In addition to
the above vectors, for example, pGEM-T, pDIRECT, and pT7 can also be used for the
subcloning and excision of cDNAs.
30 [0088]
An expression vector is especially useful for the type of vectors of the present invention.
When an expression vector is expressed, for example, in E. coli, it should have the above
characteristics in order to be amplified in E. coli. Additionally, when E. coli, such as JM109,
DH5a, HB101, or XLl-Blue are used as the host cell, the vector preferably has a promoter, for
35 example, lacZ promoter (Ward et al Nature (1989) 341 : 544-546; FASEB J. (1992) 6:
2422-2427), araB promoter (Better et al Science (1988) 240: 1041-1043), or T7 promoter, that
40 JP 2004-071763
allows efficient expression of the desired gene in E. coli. Other examples of the vectors include
pGEX-5X-l (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (where BL21, a
strain expressing T7 RNA polymerase, is preferably used as the host).
[0089]
5 Furthermore, the vector may comprise a signal sequence for polypeptide secretion.
When producing polypeptides into the periplasm of E. coli, the pelB signal sequence (Lei, S. P.
et ah J. Bacteriol. (1987) 169: 4379) may be used as a signal sequence for polypeptide secretion.
For example, calcium chloride methods or electroporation methods may be used to introduce the
vector into a host cell.
10 [0090]
In addition to E. coli, expression vectors derived from mammals (e.g., pCDNA3
(Invitrogen), pEGF-BOS (Nucleic Acids Res. (1990) 18 (17): 5322), pEF, pCDM8), insect cells
(e.g., "Bac-to-BAC baculovirus expression system" (GIBCOBRL), pBacPAK8), plants (e.g.,
pMHl, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw), retroviruses (e.g., pZIPneo),
15 yeasts (e.g., "Pichia Expression Kit" (Invitrogen), pNVll, SP-Q01), and Bacillus subtilis (e.g.,
pPL608, pKTH50) may also be used as a vector of the present invention.
[0091]
In order to express proteins in animal cells such as CHO, COS, and NIH3T3 cells, the
vector preferably has a promoter necessary for expression in such cells, for example, an SV40
20 promoter (Mulligan et ah, Nature (1979) 277: 108), MMLV-LTR promoter, EFla promoter
(Mizushima et ah, Nucleic Acids Res. (1990) 18: 5322), CMV promoter, etc.). It is even more
preferable that the vector also carries a marker gene for selecting transformants (for example, a
drug-resistance gene selected by a drug such as neomycin and G418). Examples of vectors
with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13,
25 and such.
[0092]
In addition, to stably express a gene and amplify the gene copy number in cells, CHO
cells that are defective in the nucleic acid synthesis pathway are introduced with a vector
containing a DHFR gene (for example, pCHOI) to compensate for the defect, and the copy
30 number is amplified using methotrexate (MTX). Alternatively, a COS cell, which carries an
SV40 T antigen-expressing gene on its chromosome, can be transformed with a vector
containing the S V40 replication origin (for example, pcD) for transient gene expression. The
replication origin may be derived from polyoma virus, adenovirus, bovine papilloma virus
(BPV), and such. Furthermore, to increase the gene copy number in host cells, the expression
35 vector may contain, as a selection marker, aminoglycoside transferase (APH) gene, thymidine
kinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene,
41 JP 2004-071763
dihydrofolate reductase (dhfr) gene, and such.
[0093]
Next, the vector is introduced into a host cell. The host cells into which the vector is
introduced are not particularly limited, for example, E. coli and various animal cells are available
5 for this purpose. The host cells may be used, for example, as a production system to produce
and express the antibodies of the present invention. In vitro and in vivo production systems are
available for polypeptide production systems. Production systems that use eukaryotic cells or
prokaryotic cells are examples of in vitro production systems.
[0094]
1 0 Eukaryotic cells that can be used are, for example, animal cells, plant cells, and fungal
cells. Known animal cells include: mammalian cells, for example, CHO (J. Exp. Med. (1995)
108: 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such
as Xenopus laevis oocytes (Valle, et al. } Nature (1981) 291: 358-340), or insect cells (e.g., Sf9,
Sf21, and Tn5). In the present invention, CHO-DG44, CHO-DXB11, COS7 cells, and BHK
15 cells can be suitably used. Among animal cells, CHO cells are particularly favorable for
large-scale expression. Vectors can be introduced into a host cell by, for example, calcium
phosphate methods, the DEAE-dextran methods, methods using cationic liposome DOTAP
(Boehringer-Mannheim), electroporation methods, lipofection methods.
[0095]
20 Plant cells include, for example, Nicotiana taZxzcwm-derived cells known as a protein
production system. Calluses may be cultured from these cells. Known fungal cells include
yeast cells, for example, genus Saccharomyces such as Saccharomyces cerevisiae and
Saccharomyces pombe; and filamentous fungi, for example, genus Aspergillus such as
Aspergillus niger.
25 [0096]
Bacterial cells can be used in the prokaryotic production systems. Examples of
bacterial cells include E. coli (for example, JM109, DH5<x, HB101 and such); and Bacillus
subtilis.
[0097]
30 Next, the above host cells are cultured. Antibodies can be obtained by transforming
the cells with a polynucleotide of interest and in vitro cultuiing of these transformants.
Transformants can be cultured using known methods. For example, DMEM, MEM, RPMI
1640, or IMDM may be used as the culture medium for animal cells, and may be used with or
without serum supplements such as FBS or fetal calf serum (FCS). Serum-free cultures are also
35 acceptable. The preferred pH is about 6 to 8 during the course of culturing. Incubation is
carried out typically at a temperature of about 30 to 40°C for about 15 to 200 hours. Medium is
42
JP 2004-071763
exchanged, aerated, or agitated, as necessary.
[0098]
On the other hand, production systems using animal or plant hosts may be used as
systems for producing polypeptides in vivo. For example, a polynucleotide of interest is
5 introduced into an animal or plant and the polypeptide is produced in the body of the animal or
plant and then recovered. The "hosts" of the present invention includes such animals and
plants.
[0099]
Animals to be used for the production system include mammals or insects. Mammals
10 such as goats, pigs, sheep, mice, and cattle may be used (Vicki Glaser SPECTRUM
Biotechnology Applications (1993)). Alternatively, the mammals may be transgenic animals.
[0100]
For example, a polynucleotide of interest is prepared as a fusion gene with a gene
encoding a polypeptide specifically produced in milk, such as the goat P-casein gene. DNA
15 fragments containing the fusion gene are injected into goat embryos, which are then introduced
back to female goats. The desired antibody can be obtained from milk produced by the
transgenic goats, which are bom from the goats that received the embryos, or from their
offspring. Appropriate hormones may be administered to increase the volume of milk
containing the antibody produced by the transgenic goats (Ebert, K.M. et aL, Bio/Technology
20 (1994) 12:699-702).
[0101]
Insects, such as silkworms, may also be used. Baculoviruses carrying a polynucleotide
encoding an antibody of interest can be used to infect silkworms, and the antibody of interest can
be obtained from the body fluids (Susumu, M. et al , Nature (1 985) 315: 592-594).
25 [0102]
Plants used in the production system include, for example, tobacco. When tobacco is
used, a polynucleotide encoding an antibody of interest is inserted into a plant expression vector,
for example, pMON 530, and then the vector is introduced into a bacterium, such as
Agrobacterium tumefaciens . The bacteria are then used to infect tobacco such as Nicotiana
30 tabacum, and the desired antibodies can be recovered from the leaves (Julian K.-C. Ma et al. 9 Eur.
J. Immunol. (1994) 24: 131-138).
[0103]
The resulting antibody may be isolated from the inside or outside (such as the medium)
of host cells, and purified as a substantially pure and homogenous antibody. Methods are not
35 limited to any specific method and any standard method for isolating and purifying antibodies
may be used. Polypeptides may be isolated and purified, by selecting an appropriate
43
JP 2004-071763
combination of, for example, chromatographic columns, filtration, ultrafiltration, salting out,
solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide
gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and others.
[0104]
Chromatographies include, for example, affinity chromatographies, ion exchange
chromatographies, hydrophobic chromatographies, gel filtrations, reverse-phase
chromatographies, and adsorption chromatographies (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et aL, Cold Spring Harbor
Laboratory Press, 1996). These chromatographies can be carried out using liquid phase
chromatographies such as HPLC and FPLC. Examples of the affinity chromatography columns
include protein A columns and protein G columns. Examples of the proteins A columns include
Hyper D, POROS, and Sepharose F. F. (Pharmacia).
[0105]
An antibody can be modified freely and peptide portions deleted by treating the
antibody with an appropriate protein modifying enzyme before or after antibody purification.
Such protein modifying enzymes include, for example, trypsins, chymotrypsins, lysyl
endopeptidases, protein kinases, and glucosidases.
[0106]
Antibodies that bind to Mpl can be prepared by methods known to those skilled in the
art.
For example, monoclonal antibody-producing hybridomas can be essentially generated
by known technologies as follows: immunizing animals with Mpl proteins or Mpl-expressing
cells as sensitized antigens using conventional immunological methods; fusing the obtained
immunocytes with known parental cells by conventional cell fusion methods; and screening for
monoclonal antibody-producing cells by conventional methods.
[0107]
Specifically, monoclonal antibodies can be prepared by the method below.
First, Mpl protein, which is used as a sensitized antigen for preparing antibodies, is
prepared by expressing the Mpl gene/amino acid sequence (GenBank accession number:
NM_005373). More specifically, the gene sequence encoding Mpl is inserted into a known
expression vector, which is then transfected into an appropriate host cell. The subject human
Mpl protein is purified from the host cell or culture supernatant using known methods.
[0108]
The purified Mpl protein is then used as a sensitized antigen. Alternatively, a partial
Mpl peptide may be used as a sensitized antigen. In this case, the partial peptide can also be
chemically synthesized based on the amino acid sequence of human Mpl.
44 JP 2004-071763
[0109]
The epitopes of Mpl molecule that are recognized by an anti-Mpl antibody of the
present invention are not limited to a particular epitope, and may be any epitope on the Mpl
molecule. Thus, any fragment can be used as an antigen for preparing anti-Mpl antibodies of
5 the present invention, as long as the fragment comprises an epitope of the Mpl molecule.
[0110]
There is no limitation as to the type of mammalian species to be immunized with the
sensitized antigen. However, a mammal is preferably selected based on its compatibility with
the parental cell to be used in cell fusion. Generally, rodents (for example, mice, rats, and
1 0 hamsters), rabbits, and monkeys can be used.
[0111]
Animals can be immunized with a sensitized antigen by known methods such as a
routine method of injecting a sensitized antigen into a mammal intraperitoneally or
subcutaneously. Specifically, the sensitized antigen is diluted appropriately with
1 5 phosphate-buffered saline (PBS), physiological saline and such, and then suspended. An
adequate amount of a conventional adjuvant, for example, Freund's complete adjuvant, is mixed
with the suspension, as necessary. An emulsion is then prepared for administering to a mammal
several times over a 4- to 2 1 -day interval. An appropriate carrier may be used for the sensitized
antigen in immunization.
20 [0112]
A mammal is immunized as described above. After a titer increase of target antibody
in the serum is confirmed, immunocytes are collected from the mammal and then subjected to
cell fusion. Spleen cells are the preferred immunocytes.
[0113]
25 Mammalian myeloma cells are used as the parental cells to be fused with the above
immunocytes. Preferable myeloma cells to be used include various known cell lines, for
example, P3 (P3x63Ag8.653) (Kearney JF, etal, J. Immnol. (1979) 123: 1548-1550),
P3x63Ag8U.l (Yelton DE, et aL, Current Topics in Microbiology and Immunology (1978) 81:
1-7), NS-1 (Kohler, G and Milstein, C. Eur. J. Immunol. (1976) 6: 511-519), MPC-11 (Margulies,
30 D. H. et aL, Cell (1976) 8: 405-415), SP2/0 (Shulman, M. et aL, Nature (1978) 276: 269-270),
FO (deSt. Groth, S. F. et aL, J. Immunol. Methods (1980) 35: 1-21), S194 (Trowbridge, L S., J.
Exp. Med. (1978) 148: 313-323), and R210 (Galfre, G etaL, Nature (1979) 277: 131-133).
[0114]
Cell fusions between the immunocytes and the myeloma cells as described above can be
35 essentially carried out using known methods, for example, a method by Kohler and Milstein
(Kohler, G and Milstein, C, Methods EnzymoL (1981) 73: 3-46).
45 JP 2004-071763
[0115]
More specifically, the above-described cell fusions are carried out, for example, in a
conventional culture medium in the presence of a cell fusion-promoting agent. The
fusion-promoting agents include, for example, polyethylene glycol (PEG) and Sendai virus
5 (HVJ). If required, an auxiliary substance such as dimethyl sulfoxide may also be added to
improve fusion efficiency.
[0116]
The ratio of immunocytes to myeloma cells may be determined at one's own discretion,
preferably, for example, one myeloma cell for every one to ten immunocytes. Culture media to
10 be used for the above cell fusions include, for example, media that are suitable for the growth of
the above myeloma cell lines, such as RPMI 1640 media and MEM media, and other
conventional culture media used for this type of cell culture. In addition, serum supplements
such as fetal calf serum (FCS) may also be used in combination.
[0117]
15 Cell fusion is carried out as follows. As described above, predetermined amounts of
immunocytes and myeloma cells are mixed well in the culture medium. PEG solution (for
example, mean molecular weight of about 1,000-6,000) pre-heated to 37°C is added to the cell
suspension typically at a concentration of 30% to 60% (w/v), and mixed to produce fused cells
(hybridomas). Then, an appropriate culture medium is successively added to the mixture, and
20 the sample is centrifuged to remove supernatant. This treatment is repeated several times to
remove the unwanted cell fusion-promoting agent and others that are unfavorable to hybridoma
growth.
[0118]
Screening of the resulting hybridomas can be carried out by culturing them in a
25 conventional selective medium, for example, hypoxanthine, aminopterin, and thymidine (HAT)
medium. Culturing in the above-descried HAT medium is continued for a period long enough
(typically, for several days to several weeks) to kill cells (non-fused cells) other than the desired
hybridomas. Then, hybridomas are screened for single-cell clones capable of producing the
target antibody by conventional limiting dilution methods.
30 [0119]
In addition to the method for preparing the above-descried hybridomas by immunizing
non-human animals with antigens, preferred human antibodies having binding activity to Mpl
can also be obtained by: sensitizing human lymphocytes with Mpl in vitro; and fusing the
sensitized lymphocytes with human myeloma cells capable of dividing permanently (see,
35 Japanese Patent Application Kokoku Publication No. (JP-B) H01-59878 (examined, approved
Japanese patent application published for opposition)). Alternatively, it is possible to obtain
46 JP 2004-071763
human antibodies against Mpl from immortalized cells producing anti-Mpl antibodies. In this
method, the cells producing anti-Mpl antibodies are prepared by administering Mpl as an antigen
to transgenic animals comprising a repertoire of the entire human antibody genes (see, WO
94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).
5 [0120]
The monoclonal antibody-producing hybridomas thus prepared can be passaged in a
conventional culture medium, and stored in liquid nitrogen over long periods of time.
[0121]
Monoclonal antibodies can be prepared from the above-described hybridomas by, for
1 0 example, a routine procedure of culturing the hybridomas and obtaining antibodies from the
culture supernatants. Alternatively, monoclonal antibodies can be prepared by injecting the
hybridomas into a compatible mammal; growing these hybridomas in the mammal; and
obtaining antibodies from the mammal's ascites. The former method is suitable for preparing
highly purified antibodies, while the latter is suitable for preparing antibodies on a large scale.
15 [0122]
Recombinant antibodies can also be prepared by: cloning an antibody gene from a
hybridoma; inserting the gene into an appropriate vector; introducing the vector into a host; and
producing the antibodies by using genetic recombination techniques (see, for example,
Vandamme, A. M. etal, Eur. J. Biochem. (1990) 192: 767-775).
20 [0123]
Specifically, an mRNA encoding the variable (V) region of anti-Mpl antibody is isolated
from hybridomas producing the anti-Mpl antibodies. For mRNA isolation, total RNAs are first
prepared by conventional methods such as guanidine ultracentrifixgation methods (Chirgwin, J.
M. et al, Biochemistry (1979) 18: 5294-5299), or acid guanidinium
25 thiocyanate-phenol-chloroform (AGPC) methods (Chomczynski, R et a/., Anal. Biochem. (1987)
162: 156-159), and then the target mRNA is prepared using an mRNA Purification Kit
(Pharmacia) and such. Alternatively, the mRNA can be directly prepared using the QuickPrep
mRNA Purification Kit (Pharmacia).
[0124]
30 A cDNA of the antibody V region is synthesized from the resulting mRNA using reverse
transcriptase. cDNA synthesis is carried out using the AMV Reverse Transcriptase First-strand
cDNA Synthesis Kit (Seikagaku Co.), or such. Alternatively, cDNA can be synthesized and
amplified by the 5'-RACE method (Frohman, M. A. et of., Proc. Natl. Acad. Sci. USA (1988) 85:
8998-9002; Belyavsky, A. et aL, Nucleic Acids Res. (1989) 17: 2919-2932) using the 5'-Ampli
35 FINDER RACE Kit (Clontech) and PCR.
[0125]
47
JP 2004-071763
Target DNA fragments are purified from the obtained PCR products and then ligated
with vector DNAs to prepare recombinant vectors. The vectors are introduced into E. coli and
such, and colonies are selected for preparing the recombinant vector of interest. The target
DNA nucleotide sequence is then confirmed by conventional methods such as the
5 dideoxynucleotide chain termination method.
Once a DNA encoding the V region of target anti-Mpl antibody is obtained, the DNA is
inserted into an expression vector which comprises a DNA encoding the constant region (C
region) of a desired antibody.
[0126]
10 The method for producing anti-Mpl antibodies to be used in the present invention
typically comprises the steps of: inserting an antibody gene into an expression vector, so that the
gene is expressed under the regulation of expression regulatory regions, such as enhancer and
promotor; and transforming host cells with the resulting vectors to express antibodies.
[0127]
1 5 For expressing the antibody gene, polynucleotides encoding H chain and L chain,
respectively, are inserted into separate expression vectors and co-transfected into a host cell.
Alternatively, polynucleotides encoding both H chain and L chain are inserted into a single
expression vector and transfected into a host cell (see WO 94/11523).
[0128]
20 The term "agonistic activity" refers to an activity to induce changes in some biological
activities through signal transduction into cells and such, due to the binding of an antibody to a
receptor antigen. The biological activities include, for example, proliferation-promoting
activities, proliferation activities, viability activities, differentiation-inducing activities,
differentiation activities, transcriptional activities, membrane transport activities, binding
25 activities, proteolytic activities, phosphorylation/dephosphorylation activities,
oxidation/reduction activities, transfer activities, nucleolytic activities, dehydration activities,
cell death-inducing activities, and apoptosis-inducing activities, but is not limited thereto.
[0129]
The term "agonistic activity against Mpl" typically refers to the activity of promoting
30 the differentiation of megakaryocytes or their parental hemopoietic stem cells into platelets, or
the activity of stimulating platelet proliferation.
Agonistic activity can be assayed by methods known to those skilled in the art. The
agonistic activity may be determined using the original activity or a different activity as an
indicator.
35 [0130]
For example, agonistic activity can be determined by a method using cell growth as an
48
JP 2004-071763
indicator as described in Examples. More specifically, an antibody whose agonistic activity is
to be determined is added to cells which proliferate in an agonist-dependent manner, followed by
incubation of the cells. Then, a reagent such as WST-8 which shows a coloring reaction at
specific wavelengths depending on the viable cell count, is added to the culture and absorbance
5 is measured. The agonistic activity can be determined using the measured absorbance as an
indicator.
[0131]
Cells that proliferate in an agonist-dependent manner can also be prepared by methods
known to those skilled in the art. For example, when the antigen is a receptor capable of
1 0 transducing cell growth signals, cells expressing the receptor may be used. Alternatively, when
the antigen is a receptor that cannot transduce signals, a chimeric receptor consisting of the
intracellular domain of a receptor that transduces cell growth signals and the extracellular
domain of a receptor that does not transduce cell growth signals can be prepared for cellular
expression. Receptors that transduce cell growth signals include, for example, G-CSF receptors,
15 mpl, neu, GM-CSF receptors, EPO receptors, c-kit, and FLT-3. Cells that can be used to
express a receptor include, for example, BaF3, NFS60, FDCP-1, FDCP-2, CTLL-2, DA-1, and
KT-3.
[0132]
There is no limitation as to the type of detection indicators to be used for determining
20 agonistic activity, as long as the indicator can monitor quantitative and/or qualitative changes.
For example, it is possible to use cell-free assay indicators, cell-based assay indicators,
tissue-based assay indicators, and in vivo assay indicators. Indicators that can be used in
cell-free assays include enzymatic reactions, quantitative and/or qualitative changes in proteins,
DNAs, or RNAs. Such enzymatic reactions include, for example, amino acid transfers, sugar
25 transfers, dehydrations, dehydrogenations, and substrate cleavages. Alternatively, protein
phosphorylations, dephosphorylations, dimerizations, multimerizations, hydrolyses, dissociations
and such; DNA or RNA amplifications, cleavages, and extensions can be used as the indicator in
cell-free assays. For example, protein phosphorylations downstream of a signal transduction
pathway may be used as a detection indicator. Alterations in cell phenotype, for example,
30 quantitative and/or qualitative alterations in products, alterations in growth activity, alterations in
cell number, morphological alterations, or alterations in cellular properties, can be used as the
indicator in cell-based assays. The products include, for example, secretory proteins, surface
antigens, intracellular proteins, and mRNAs. The morphological alterations include, for
example, alterations in dendrite formation and/or dendrite number, alteration in cell flatness,
35 alteration in cell elongation/axial ratio, alterations in cell size, alterations in intracellular
structure, heterogeneity/homogeneity of cell populations, and alterations in cell density. Such
49 JP 2004-071763
morphological alterations can be observed under a microscope. Cellular properties to be used
as the indicator include anchor dependency, cytokine-dependent response, hormone dependency,
drug resistance, cell motility, cell migration activity, pulsatory activity, and alteration in
intracellular substances. Cell motility includes cell infiltration activity and cell migration
5 activity. The alterations in intracellular substances include, for example, alterations in enzyme
activity, mRNA levels, levels of intracellular signaling molecules such as Ca 2+ and cAMP, and
intracellular protein levels. When a cell membrane receptor is used, alterations in the cell
proliferating activity induced by receptor stimulation can be used as the indicator. The
indicators to be used in tissue-based assays include functional alterations adequate for the subject
10 tissue. In in vivo assays, alterations in tissue weight, alterations in the blood system (for
example, alterations in blood cell counts, protein contents, or enzyme activities), alterations in
electrolyte levels, and alterations in the circulating system (for example, alterations in blood
pressure or heart rate).
[0133]
1 5 The methods for measuring such detection indices are not particularly limited. For
example, absorbance, luminescence, color development, fluorescence, radioactivity, fluorescence
polarization, surface plasmon resonance signal, time-resolved fluorescence, mass, absorption
spectrum, light scattering, and fluorescence resonance energy transfer may be used. These
measurement methods are known to those skilled in the art and may be selected appropriately
20 depending on the purpose. For example, absorption spectra can be obtained by using a
conventional photometer, plate reader, or such; luminescence can be measured with a
luminometer or such; and fluorescence can be measured with a fluorometer or such. Mass can
be determined with a mass spectrometer. Radioactivity can be determined with a device such
as a gamma counter depending on the type of radiation. Fluorescence polarization can be
25 measured with BEACON (TaKaRa). Surface plasmon resonance signals can be obtained with
BIACORE. Time-resolved fluorescence, fluorescence resonance energy transfer, or such can
be measured with ARVO or such. Furthermore, a flow cytometer can also be used for
measuring. It is possible to use one of the above methods to measure two or more different
types of detection indices. A greater number of detection indices may also be examined by
30 using two or more measurement methods simultaneously and/or consecutively. For example,
fluorescence and fluorescence resonance energy transfer can be measured at the same time with a
fluorometer.
[0134]
The present invention provides pharmaceutical compositions comprising antibodies of
35 this invention. The pharmaceutical compositions comprising antibodies of the present
invention are useful for treating and/or preventing thrombocytopenia and such. Time required
50
JP 2004-071763
for the platelet count to recover to the normal level can be shortened by administering an
antibody of the present invention after donation of platelet components. The amount of platelet
components at the time of blood collection can be increased by pre-administering an antibody of
the present invention.
5 [0135]
When used as pharmaceutical compositions, the antibodies of the present invention can
be formulated by methods known to those skilled in the art. For example, the antibodies can be
administered parenterally by injection of a sterile solution or suspension in water or other
pharmaceutically acceptable solvents. For example, the antibodies can be formulated by
1 0 appropriately combining with pharmaceutically-acceptable carriers or solvents, specifically,
sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants,
stabilizers, flavoring agents, excipients, vehicles, preservatives, binding agents, and such, and
mixing at a unit dosage and form required by accepted pharmaceutical implementations. In
such formulations, the amount of the thus obtained active ingredient should be within the
1 5 required range .
[0136]
A sterile composition to be injected can be formulated using a vehicle such as distilled
water used for injection, according to standard protocols.
Aqueous solutions used for injections include, for example, physiological saline and
20 isotonic solutions comprising glucose or other adjunctive agents such as D-sorbitol, D-mannose,
D-mannitol, and sodium chloride. They may also be combined with an appropriate solubilizing
agent such as alcohol, specifically, ethanol, polyalcohol such as propylene glycol or polyethylene
glycol, or non-ionic detergent such as polysorbate 80™ or HCO-50, as necessary.
[0137]
25 Oil solutions include sesame oils and soybean oils, and can be combined with
solubilizing agents such as benzyl benzoate or benzyl alcohol. Injection solutions may also be
formulated with buffers, for example, phosphate buffers or sodium acetate buffers; analgesics,
for example, procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or
anti-oxidants. The prepared injections are typically aliquoted into appropriate ampules.
30 [0138]
The administration is preferably carried out parenterally, specifically, by injection,
intranasal administration, intrapulmonary administration, percutaneous administration, or such.
Injections include, for example, intravenous injections, intramuscular injections, intraperitoneal
injections, and subcutaneous injections. The injection solutions can be also administered
35 systemically or locally.
[0139]
51
JP 2004-071763
The administration methods can be selected properly according to the patient's age,
condition, and such. The applied dose of a pharmaceutical composition comprising an antibody
or polynucleotide encoding the antibody may be, for example, in the range of 0.0001 to 1,000
mg/kg body weight. Alternatively, the dosage may be, for example, in the range of 0.001 to
5 100,000 mg/kg body weight. However, the dosage is not restricted to the values described
above. The dosage and administration methods depend on the patient's weight, age, and
condition, and are appropriately selected by those skilled in the art.
[Examples]
[0140]
10 The present invention is specifically illustrated below with reference to Examples, but it
is not to be construed as being limited thereto.
[0141]
[Example 1] Preparation of anti-human Mpl antibodies
1.1 Establishment of Mpl-expressing BaF3 cell lines
15 BaF3 cell lines expressing the full-length Mpl gene were established to obtain cell lines
that proliferate in a TPO-dependent manner.
A full-length human Mpl cDNA (Palacios, R. et ai, Cell (1985) 41 : 727-734) (GenBank
accession NO. NM_005373) was amplified by PCR. The cDNA was cloned into a pCOS2
expression vector to construct pCOS2-hMplfull. The expression vector pCOS2 was
20 constructed by removing the DHFR gene expression region from pCHOI (Hirata, Y. et ai, FEBS
Letter (1994) 356: 244-248), where the expression region of the neomycin resistance gene
HEF-VH-gyl (Sato, K. et al, Mol Immunol. (1994) 31: 371-381) is inserted.
[0142]
The cynomolgus monkey Mpl cDNA (SEQ ID NO: 164) was cloned from total RNA
25 extracted from the bone marrow cells of cynomolgus monkey, using a SMART RACE cDNA
Amplification Kit (Clontech). The resulting cynomolgus monkey cDNA was. inserted into
pCOS2 to construct pCOS2-monkeyMplfull.
Then, the full-length mouse Mpl cDNA (GenBank accession NO. NM_0 10823) was
amplified by PCR, and inserted into pCOS2 to construct pCOS2-mouseMplfull.
30 [0143]
Each vector (20 jig) prepared as described above was mixed with BaF3 cells (1 x 10 7
cells/mL) suspended in PBS in Gene Pulser cuvettes. This mixture was then pulsed at 0.33 kV
and 950 jxFD using a Gene Pulser II (Bio-Rad). The BaF3 cells introduced with the above
DNAs by electroporation were added to RPMI 1640 medium (Invitrogen) containing 1 ng/mL
35 mouse interleukin 3 (hereinafter abbreviated as mIL-3; Peprotech), 500 ng/mL Geneticin
(Invitrogen), and 10% FBS (Invitrogen), and selected to establish a human Mpl-expressing BaF3
JP 2004-071763
cell line (hereinafter abbreviated as "BaF3-human Mpl"), monkey Mpl-expressing BaF3 cell line
(hereinafter abbreviated as BaF3 -monkey Mpl), and mouse Mpl-expressing BaF3 cell line
(hereinafter abbreviated as "BaF3 -mouse Mpl"). Following selection, these cells were cultured
and maintained in RPMI 1640 containing 1 ng/mL rhTPO (R&D) and 10% FBS.
5 [0144]
1 .2 Establishment of Mpl-expressing CHO cell lines
CHO cell lines expressing the full-length Mpl gene were established to obtain cell lines
to be used for assessing binding activity by flow cytometry.
First, the DHFR gene expression site from pCHOI was inserted into pCXN2 (Niwa, H.
1 0 et al , Gene ( 1 99 1 ) 1 08 : 1 93 - 1 99) at the Hindlll site to prepare a pCXND3 expression vector.
The respective Mpl genes were amplified by PCR using pCOS2-hMplfull,
pCOS2-monkeyMplfull, and pCOS2-mouseMplfiill as templates, and primers with a His-tag
sequence. The PCR products were cloned into pCXND3 to construct pCXND3-hMpl-His,
pCXND3-monkey Mpl-His, and pCXND3 -mouse Mpl-His, respectively.
15 [0145]
Vectors thus prepared (25 |j,g each) were mixed with a PBS suspension of CHO-DG44
7 -
cells (1x10 cells/mL) in Gene Pulser cuvettes. The mixture was then pulsed at 1 .5 kV and 25
^iFD using Gene Pulser II (Bio-Rad). The CHO cells introduced with these DNAs by
electroporation were added to CHO-S-SFMII medium (Invitrogen) containing 500 p,g/mL
20 Geneticin and lx HT (Invitrogen). A human Mpl-expressing CHO cell line (hereinafter
abbreviated as "CHO-human Mpl"), monkey Mpl-expressing CHO cell line (hereinafter
abbreviated as "CHO-monkey Mpl"), and mouse Mpl-expressing CHO cell line (hereinafter
abbreviated as "CHO-mouse Mpl") were established through selection.
[0146]
25 1 .3 Preparation of soluble human Mpl protein
To prepare soluble human Mpl protein, an expression system using insect Sf9 cells for
production and secretion of the protein was constructed as described below.
A DNA construct encoding the extracellular region of human Mpl (Gin 26 to Tip 491)
with a downstream FLAG tag was prepared. The construct was inserted into a pBACSurf-1
30 Transfer Plasmid (Novagen) between the Pstl and Smal sites to prepare pBACSurfl-hMpl-FLAG
Then, Sf9 cells were transformed with 4 jig of pBACSurfl-hMpl-FLAG using the Bac-N-Blue
Transfection Kit (Invitrogen). The culture supernatant was collected after three-day incubation.
Recombinant virus was isolated by plaque assays. The prepared virus stock was used to infect
Sf9 cells, and the culture supernatant was collected.
35 [0147]
Soluble human Mpl protein was purified from the obtained culture supernatant as
53
JP 2004-071763
described below. The culture supernatant was loaded onto a Q Sepharose Fast Flow
(Amersham Biosciences) for adsorption, and the adsorbed protein was then eluted with 50 mM
Na-phosphate buffer (pH7.2) containing 0.01% (v/v) Tween20 and 500 mM NaCl. After the
eluates were loaded onto a FLAG M2 -Agarose (Sigma- Aldrich) for adsorption, the protein
5 adsorbed was eluted with 100 mM glycine-HCl buffer (pH3.5) containing 0.01% (v/v) Tween20.
Immediately after elution, the fraction obtained was neutralized with 1 M Tris-Cl (pH8.0) and
the buffer was exchanged with PBS (-) and 0.01% (v/v) Tween20 using PD-10 columns
(Amersham Biosciences). The purified soluble Mpl protein was referred to as "shMpl-FLAG".
[0148]
10 1 .4 Preparation of human Mpl-IgG Fc fusion protein
Human fusion protein Mpl-IgG Fc gene was prepared according to the method by
Bennett et al (Bennett, B. D. etal.J. Biol. Chem. (1991) 266: 23060-23067). Anucleotide
sequence encoding the extracellular region of human Mpl (Gin 26 to Trp 491) was linked to a
nucleotide sequence encoding the Fc region of human IgG-yl (a region downstream of Asp 216).
15 ABstEll sequence (amino acids: Val-Thr) was attached to the junction as a fusion linker between
these two regions. A 19-amino acid signal peptide derived form human IgG H chain variable
region was used as the signal sequence. The resulting human fusion protein Mpl-IgG Fc gene
was cloned into pCXND3 to construct pCXND3 -hMpl-Fc.
[0149]
20 The vector thus prepared (25 jag) was mixed with a PBS suspension of CHO-DG44 cells
(1x10 cells/mL) in Gene Pulser cuvettes. The mixture was then pulsed at 1.5 kV and 25 \i¥D
using Gene Pulser II (Bio-Rad). The CHO cells introduced with the DNA by electroporation
were added to CHO-S-SFMII medium containing 500 ^ig/mL Geneticin and lx HT (Invitrogen).
shMPL-Fc-expressing CHO cell line (CHO-hMpl-Fc) was then established through selection.
25 [0150]
Human Mpl-IgG Fc fusion protein was purified from the culture supernatant as
described below.
The culture supernatant was loaded onto a Q Sepharose Fast Flow (Amersham
Biosciences) for adsorption, and then the adsorbed protein were eluted with 50 mM
30 Na-phosphate buffer (pH7.6) containing 0.01% (v/v) Tween20 and 1 M NaCl. After the eluates
were loaded onto a HiTrap protein G HP column (Amersham Biosciences) for adsorption, the
adsorbed protein was eluted with 0. 1 M glycine-HCl buffer (pH2.7) containing 150 mM NaCl
and 0.01% (v/v) Tween20. Immediately after elution, the obtained fraction was neutralized
with 1 M Tris-Cl (pH8.0) and the buffer was exchanged with PBS (-) and 0.01% (v/v) Tween20
35 using PD-10 columns (Amersham Biosciences). The purified soluble Mpl protein was referred
to as "hMpl-Fc".
54
JP 2004-071763
[0151]
1.5 Immunization with shMpl-FLAG or BaF3 -human Mpl and hybridoma selection
MRL/MpJUmmCrj-lpr/lpr mice (hereinafter abbreviated as "MRL/lpr mice"; purchased
from Charles River, Japan) were immunized; the primary immunization was carried out at eight
5 weeks of age. For every single mouse, an emulsion containing 1 00 |Lig of shMPL-FLAG
combined with Freund's complete adjuvant (H37 Ra; Beckton Dickinson), was administered
subcutaneously as the primary injection. As a booster injection, an emulsion containing
shMPL-FLAG (50 ng per mouse) combined with Freund's incomplete adjuvant (Beckton
Dickinson) was administered subcutaneously. . Three mice which have been immunized six
10 times in total were subjected to a final injection of shMPL-FLAG (50 \ig per mouse) through the
caudal vein. Cell fusion was achieved by mixing the mouse myeloma P3-X63Ag8Ul cells
(P3U1; purchased from ATCC) and mouse splenocytes using polyethylene glycol 1500 (Roche
Diagnostics). Hybridoma selection in HAT medium began the following day and culture
supernatants were obtained. Screening was carried out by ELISA, using immunoplates
1 5 immobilized with shMpl-FLAG or hMpl-Fc and the assayed cell growth activity of BaF3 -human
Mpl as an index. In addition, Balb/C mice were immunized eleven times in total by
administering BaF3-human Mpl (1 .0 x 10 cells per mouse) intraperitoneally over a period of
one week to five months. Hybridomas were similarly prepared by cell fusion, and screened
using the assayed cell growth activity of BaF3 -human Mpl as an index. Positive clones were
20 isolated as single clones by limiting dilution and then cultured in a large scale. The culture
supernatants were collected.
[0152]
1 .6 Analyses of anti-human Mpl antibodies
Antibody concentrations were determined by carrying out a mouse IgG sandwich
25 ELISA using goat anti-mouse IgG (gamma) (ZYMED) and alkaline phosphatase- goat
anti-mouse IgG (gamma) (ZYMED), generating a calibration curve by GraphPad Prism
(GraphPad Software; USA), and calculating the antibody concentrations from the calibration
curve. Commercially available antibodies of the same isotype were used as standards.
[0153]
30 Antibody isotypes were determined by antigen-dependent ELISA using isotype-specific
secondary antibodies. hMpl-Fc was diluted to 1 ng/mL with a coating buffer (0.1 mM NaHCC>3,
pH9.6) containing 0.02% (w/v) NaN 3 , and then added to ELISA plates. The plates were
incubated overnight at 4°C for coating. The plates were blocked with a diluent buffer (50 mM
Tris-HCl (pH8.1) containing 1 mM MgCl 2 , 150 mM NaCl, 0.05% (v/v) Tween20, 0.02% (w/v)
35 NaN3, 1% (w/v) BSA). After the addition of hybridoma culture supernatants, the plates were
allowed to stand at room temperature for 1 hr. After washing with a rinse buffer (0.05% (v/v)
55
JP 2004-071763
Tween20 in PBS), alkaline phosphatase-labeled isotype-specific secondary antibodies were
added to the plates. Then, the plates were allowed to stand at room temperature for 1 hr.
Color development was carried out using SIGMA 104 (Sigma- Aldrich) diluted to 1 mg/mL with
a substrate buffer (50 mM NaHCC>3, pH9.8) containing 10 mM MgCk, and absorbance was
5 measured at 405 nm using Benchmark Plus (Bio-Rad).
[0154]
The binding activities of an antibody to shMpl-FLAG and hMPL-Fc were determined
by ELISA. ELIS A plates were coated with 1 (ag/mL of purified shMpl-FLAG or hMPL-Fc, and
blocked with a diluent buffer. Hybridoma culture supernatants were added to the plates, and the
10 plates were allowed to stand at room temperature for 1 hr. Then, alkaline phosphatase-labeled
anti-mouse IgG antibodies (Zymed) were added to the plates. Color development was similarly
carried out using the above method. Following a one-hour coloring reaction at room
temperature, absorbance was measured at 405 nm and EC50 values were computed using
GraphPad Prism.
15 [0155]
CHO-human Mpl cells and CHO-monkey Mpl cells were harvested, and suspended in
FACS Buffer (1% FBS/ PBS) to a final concentration of 1 x 10 6 cells/mL. The suspensions
were aliquoted into Multiscreen (Millipore) at 100 fil/well, and the culture supernatants were
removed by centrifugation. Culture supernatants diluted to 5 jig/mL were added to the plates
20 and incubated on ice for 30 min. The cells were washed once with FACS buffer, and incubated
on ice for 30 min following the addition of an FITC-labeled anti-mouse IgG antibody (Beckman
Coulter). After incubation, the mixture was centrifuged at 500 rpm for 1 min. The
supernatants were removed, and then the cells were suspended in 400 jiL of FACS buffer. The
samples were analyzed by flow cytometry using EPICS ELITE ESP (Beckman Coulter). An
25 analysis gate was set on the forward and side scatters of a histogram to include viable cell
populations.
[0156]
Agonistic activities of an antibody were evaluated using BaF3 -human Mpl and
BaF3-monkey Mpl which proliferate in a TPO-dependent manner. Cells of each cell line were
30 suspended at 4 x 10 5 cells/ml in RPMI 1640/10% FBS (Invitrogen), and each suspension was
aliquoted into a 96-well plate at 60|al/well. A 40-jj.L aliquot of rhTPO (R&D) and hybridoma
culture supernatants prepared at various concentrations was added into each well. The plates
were then incubated at 37°C under 5% C0 2 for 24 hr. A 10-jaL aliquot of the Cell Count
Reagent SF (Nacalai Tesque) was added into each well. After incubation for 2 hr, absorbance
35 was measured at 450 nm (and at 655 nm as a control) using a Benchmark Plus. EC50 values
were calculated using GraphPad Prism.
56 JP 2004-071763
The above analysis yielded a total of 163 clones of mouse monoclonal antibodies that
bind to human Mpl.
Among the anti-human Mpl antibodies to be described, TA136 was established from
mice immunized with BaF-human Mpl and the others were established from mice immunized
5 with shMpl-Flag.
[0157]
1.7 Purification of anti-human Mpl antibodies
Anti-human Mpl antibodies were purified from hybridoma culture supernatants as
described below.
1 0 After the culture supernatants were loaded onto HiTrap protein G HP columns
(Amersham Biosciences) for adsorption, the antibodies were eluted with 0.1 M glycine-HCl
(pH2.7). Immediately after elution, the fractions were neutralized with 1 M Tris-Cl (pH9.0),
dialyzed against PBS for one day, and the buffer was replaced.
[0158]
15 1.8 Determination of epitopes for the anti-human Mpl antibody VB22B
Since the anti-human Mpl antibody VB22B can be used for Western blotting, a
GST-fusion protein containing a partial sequence of human Mpl was constructed for VB22B
epitope analysis. MG1 (Gln26 to Trp491) and MG2 (Gln26 to Leu274) regions were each
amplified by PCR, and cloned into pGEX-4T-3 (Amersham Biosciences) to be expressed as GST
20 fusion proteins. The resulting plasmid DNAs were transformed into DH5a to give
transformants. A final concentration of 1 mM IPTG was added to the transformants in their
logarithmic growth phase to induce the expression of GST fusion proteins. The bacterial cells
were harvested after two hours of incubation. The cells were lysed by sonication. The lysates
were centrifuged in XL-80 Ultracentrifuge (Beckman, Rotor 70.1Ti) at 35,000 rpm for 30 min.
25 The culture supernatants were removed, and then the fusion proteins were purified using GST
Purification Modules (Amersham Biosciences). The samples were separated by
10%-SDS-PAGE, and then transferred onto a PVDF membrane. The membrane was Western
blotted with the murine antibody VB22B. VB22B was found to recognize both MG-1 and
MG-2, indicating that the VB22B epitope is located in the (Gln26 to Leu274) region.
30 [0159]
Then, GST fusion proteins containing the respective regions of human Mpl: MG3
(Gln26 to Alal 89), MG4 (Gln26 to Prol06), MG5 (Gln26 to Glu259), and MG6 (Gln26 to
Gly245) were prepared and analyzed by Western blotting using the same procedure described
above. VB22B was found to recognize MG5 and MG6, but not MG3 and MG4. This
35 suggests that the VB22B epitope is located within the (Alal 89 to Gly245) region. In addition,
GST was fused with MG7 (Gln26 to Ala231) and MG8 (Gln26 to Pro217) to prepare GST fusion
57
JP 2004-071763
proteins. VB22B recognized MG7 but not MG8, suggesting that the VB22B epitope is located
in the (Gln2 1 7 to Ala23 1 ) region. Furthermore, GST fusion protein containing MG 1 0 (Gln2 1 3
to Ala231) was recognized by VB22B, suggesting that the VB22B epitope is located within the
limited region of 19 amino acids between Gln213 and Ala231.
5 [0160]
1 .9 Kinetic analyses of the antigen-antibody reaction for anti-human Mpl antibody VB22B
Since the anti-human Mpl antibody VB22B binds to soluble recombinant Mpl, kinetic
analyses of the antigen-antibody reaction between VB22B IgG and human Mpl-IgG Fc fusion
protein were carried out as described in Example 1 .4. The Sensor Chip CMS (Biacore) was
10 placed in Biacore 2000 (Biacore), and human Mpl-IgG Fc fusion protein was immobilized onto
the chip by amine-coupling methods. Then, 1.25 to 20 |j,g/mL of VB22B IgG solution was
prepared using HBS-EP Buffer (Biacore), and injected over the chip surface for 2 min to reveal
the binding region. Then, HBS-EP Buffer was injected over the chip surface for 2 min to reveal
the dissociation region. VB22B IgG bound to the human Mpl-IgG Fc fusion protein on the
15 sensor chip was removed by injecting 10 mM NaOH over the sensor chip for 15 sec, and the chip
was recovered. HBS-EP Buffer was used as the running buffer, and the flow rate was 20
|iL/min. Using the BIAevaluation Version 3.1 (Biacore) software, the reaction rate constant at
each concentration was calculated from the sensorgrams. The dissociation constant (KD) for
VB22B IgG was determined to be 1.67 ± 0.713 x 10" 9 M.
20 [0161]
[Example 2] Preparation of single-chain anti-human Mpl antibodies
Among the prepared anti-human Mpl antibodies, 23 types of antibodies, which exhibit
higher binding activities and agonistic activities, were selected to construct expression systems
for single-chain antibodies using genetic engineering techniques. An exemplary method for
25 constructing a single-chain antibody derived from the anti-human Mpl antibody VB22B is
described below.
[0162]
2.1 Cloning of the anti-human Mpl antibody variable region
The variable region was amplified by RT-PCR using total RNA extracted from
30 hybridomas producing anti-human Mpl antibodies. Total RNA was extracted from 1x10
hybridoma cells using the RNeasy Plant Mini Kit (QIAGEN).
[0163]
A 5 5 -terminal fragment of the gene was amplified from 1 jag of total RNA by the
SMART RACE cDNA Amplification Kit (Clontech), using a synthetic oligonucleotide
35 MHC-IgG2b (SEQ ID NO: 166) complementary to mouse IgG2b constant region or a synthetic
oligonucleotide kappa (SEQ ID NO: 167) complementary to mouse k chain constant region.
58 JP 2004-071763
Reverse transcription was carried out at 42°C for 1.5 hr.
[0164]
The composition of the PCR reaction solution (50 (J.L in total) is shown below.
1 Ox Advantage 2 PCR Buffer (Clontech) 5 (iL
1 Ox Universal Primer A Mix (Clontech) 5 jaL
dNTPs (dATP, dGTP, dCTP, and dTTP) (Clontech) 0.2 mM
Advantage 2 Polymerase Mix (Clontech) 1 jliL
Reverse transcription product 2.5 |liL
Synthetic oligonucleotide, MHC-IgG2b or kappa 10 pmol
[0165]
5 The PCR reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 5 sec and 72°C for 3 min;
five cycles of 94°C for 5 sec, 70°C for 10 sec, and 72°C for 3 min;
25 cycles of 94°C for 5 sec, 68°C for 10 sec, and 72°C for 3 min;
1 0 and final extension was at 72°C for 7 min.
[0166]
The PCR products were purified from agarose gel using the QIAquick Gel Extraction
Kit (QIAGEN), and cloned into a pGEM-T Easy Vector (Promega). The nucleotide sequence
was then determined using the ABI 3700 DNA Analyzer (Perkin Elmer).
15 [0167]
The nucleotide sequence of cloned VB22B H chain variable region (hereinafter
abbreviated as "VB22B-VH") is shown in SEQ ID NO: 117, and its amino acid sequence is
shown in SEQ ID NO: 118. The nucleotide sequence of the L chain variable region (hereinafter
abbreviated as "VB22B-VL") is shown in SEQ ID NO: 119, and its amino acid sequence is
20 shown in SEQ ID NO: 120.
[0168]
2.2 Preparation of expression vectors for anti-human Mpl diabodies
The gene encoding VB22B single-chain Fv (hereinafter abbreviated as "VB22B
diabody") containing a five-amino acid linker sequence was constructed, by linking a nucleotide
25 sequence encoding a (Gly4Ser)i linker to the VB22B-VH-encoding gene at its 3' end and to the
VB22B-VL-encoding gene at its 5 ' end; both of which have been amplified by PCR.
[0169]
The VB22B-VH forward primer, 70115HF, (SEQ ID NO: 168) was designed to contain
an EcoRI site. The VB22B-VH reverse primer, 33415HR, (SEQ ID NO: 169) was designed to
30 hybridize to a DNA encoding the C terminus of VB22B-VH, and to have a nucleotide sequence
59 JP 2004-071763
encoding the (Gly4Ser)i linker and a nucleotide sequence hybridizing to the DNA encoding the
N terminus of VB22B-VL. The VB22B-VL forward primer, 33-11 5LF, (SEQ ID NO: 1 70) was
designed to have a nucleotide sequence encoding the N terminus of VB22B-VL, a nucleotide
sequence encoding the (Gly4Ser)i linker, and a nucleotide sequence encoding the C terminus of
5 VB22B-VH. The VB22B-VL reverse primer, 33-115LR, (SEQ ID NO: 171) was designed to
hybridize to a DNA encoding the C terminus of VB22B-VL and to have a nucleotide sequence
encoding a FLAG tag (Asp Tyr Lys Asp Asp Asp Asp Lys/SEQ ID NO: 172) and a Notl site.
[0170]
In the first round of PCR, two PCR products: one containing VB22B-VH and a linker
1 0 sequence, and the other containing VB22B- VL and the identical linker sequence, were
synthesized by the procedure described below.
The composition of the PCR reaction solution (50 jaL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5 \\L
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
pGEM-T Easy vector comprising VB22B- VH or VB22B- VL gene 1 0 ng
Synthetic oligonucleotides, 70-115HF and 33115HR, or 33415LF lOpmol
and33115LR
[0171]
The PCR reaction conditions were:
15 94°C (initial temperature) for 30 sec;
five cycles of: 94°C for 15 sec and 72°C for 2 min;
five cycles of 94°C for 15 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
20 [0172]
After the PCR products of about 400 bp were purified from agarose gel using the
QIAquick Gel Extraction Kit (QIAGEN), the second-round PCR was carried out using aliquots
of the respective PCR products according to the protocol described below.
The composition of the PCR reaction solution (50 \iL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5 nL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 unit
First-round PCR products (two types) 1 jjJL
Synthetic oligonucleotides, 70115HF and 33-1 15LR 10 pmol
25 [0173]
60 JP 2004-071763
The reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 1 5 sec and 72°C for 2 min;
five cycles of 94°C for 15 sec and 70°C for 2 min;
5 28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0174]
The PCR products of about 800 bp were purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN), and then digested with EcoRI and Notl (both from TaKaRa).
10 The resulting DNA fragments were purified using the QIAquick PCR Purification Kit
(QIAGEN), and then cloned into pCXND3 to prepare pCXND3-VB22B db.
[0175]
2.3 Preparation of expression vectors for anti-human Mpl antibody sc(Fv)2
To prepare expression plasmids for the modified antibody [sc(Fv)2] comprising two
1 5 units of H chain variable region and two units of L chain variable region derived from VB22B,
the above-described pCXND3-VB22B db was modified by PCR using the procedure shown
below. The process for constructing the sc(Fv) 2 gene is illustrated in Fig. 1 .
[0176]
First, PCR method was carried out to amplify (a) the VB22B-VH-encoding gene in
20 which a nucleotide sequence encoding a 15-amino acid linker (Gly4Ser)3 was added to its 3' end;
and (b) the VB22B-VL-encoding gene containing the identical linker nucleotide sequence added
to its 5' end. The desired construct was prepared by linking these amplified genes. Three new
primers were designed in this construction process. The VB22B-VH forward primer,
VB22B-fpvu, (primer A; SEQ ID NO: 173) was designed to have an EcoKL site at its 5 5 end and
25 to convert Gln22 and Leu23 of VB22B db into a Pvull site. The VB22B-VH reverse primer,
sc-rL15, (primer B; SEQ ID NO: 174) was designed to hybridize to a DNA encoding the C
terminus of VB22B-VH, and to have a nucleotide sequence encoding the (Gly4Ser) 3 linker, as
well as a nucleotide sequence hybridizing to a DNA encoding the N terminus of VB22B-VL.
The VB22B-VL forward primer, sc-fL15, (primer C; SEQ ID NO: 175) was designed to have a
30 nucleotide sequence encoding the N terminus of VB22B-VL, a nucleotide sequence encoding the
(Gly4Ser)3 linker, and a nucleotide sequence encoding the C terminus of VB22B-VH.
[0177]
In the first-round PCR, two PCR products: one comprising VB22B-VH and a linker
sequence, and the other comprising VB22B-VL and the identical linker sequence, were
35 synthesized by the procedure described below.
The composition of the PCR reaction solution (50 \xL in total) is shown below.
61 JP 2004-071763
lOxPCR Buffer (TaKaRa) 5
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
pCXND3-VB22B db 10 ng
Synthetic oligonucleotides, VB22B-fpvu, sc-rL15 or sc-fL15, and lOpmol
33- 115LR (primer D)
[0178]
The reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 15 sec and 72°C for 2 min;
5 five cycles of 94°C for 1 5 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0179]
After the PCR products of about 400 bp were purified from agarose gel using the
1 0 QIAquick Gel Extraction Kit (QIAGEN), the second-round PCR was carried out using aliquots
of the respective PCR products according to the protocol described below.
The composition of the PCR reaction solution (50 uL in total) is shown below.
lOx PCR Buffer (TaKaRa) 5 uL
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
First-round PCR product (two types) 1 uL
Synthetic oligonucleotide, 70115HF and 33115LR 10 pmol
[0180]
The reaction conditions were:
15 94°C (initial temperature) for 30 sec;
five cycles of 94°C for 1 5 sec and 72°C for 2 min;
five cycles of 94°C for 15 sec and 70°C for 2 min;
28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
20 [0181]
The PCR products of about 800 bp were purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN), and then digested with EcoRI and Notl (both from TaKaRa).
The resulting DNA fragments were purified using the QIAquick PCR Purification Kit
(QIAGEN), and then cloned into pBacPAK9 (Clontech) to construct pBacPAK9-scVB22B.
25 [0182]
62
JP 2004-071763
A fragment to be inserted into the Pvull site of pBacPAK9-scVB22B was prepared.
Specifically, the fragment has a Pvull recognition site at both ends and a nucleotide sequence, in
which a gene encoding the VB22B-VH N-terminus is linked, via a (Gly4Ser)3 linker-encoding
nucleotide sequence, to a gene encoding the amino acid sequence of an N-terminus-deleted
5 VB22B-VH linked to VB22B-VL via the (Gly4Ser) 3 linker. Two primers were newly designed
to prepare the fragment by PCR. The forward primer for the fragment of interest, Fv2-f (primer
E; SEQ ID NO: 176), was designed to have a PvwII site at its 5' end and a VB22B-VH 5 5 -end
sequence. The reverse primer for the fragment of interest, Fv2-r (primer F; SEQ ID NO: 177),
was designed to hybridize to a DNA encoding the C terminus of VB22B-VL, and to have a Pvull
10 site, a nucleotide sequence encoding the (Gly4Ser)3 linker, and a nucleotide sequence hybridizing
to a DNA encoding the N terminus of VB22B-VH. PCR was carried out using
pBacPAK9-scVB22B as a template as described below.
[0183]
The composition of the PCR reaction solution (50 |iL in total) is shown below.
1 Ox PCR Buffer (TaKaRa) 5
dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 0.4 mM
DNA polymerase TaKaRa Ex Taq (TaKaRa) 2.5 units
pBacPAK9-sc VB22B 1 0 jig
Synthetic oligonucleotide, Fv2-f and Fv2-r 1 0 pmol
15 [0184]
The reaction conditions were:
94°C (initial temperature) for 30 sec;
five cycles of 94°C for 15 sec and 72°C for 2 min;
five cycles of 94°C for 1 5 sec and 70°C for 2 min;
20 28 cycles of 94°C for 15 sec and 68°C for 2 min;
and final extension was at 72°C for 5 min.
[0185]
The PCR products of about 800 bp were purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN), and then cloned into the pGEM-T Easy Vector (Promega).
25 After sequencing, the plasmid was digested with Pvull (TaKaRa), and the fragment of interest
was recovered. The recovered fragment was ligated to pBacPAK9-scVB22B pre-digested with
PvwII (TaKaRa) to construct pBacPAK9-VB22B sc(Fv) 2 . After the resulting vector was
digested with EcoRI and Notl (both from TaKaRa), the fragment of about 1,600 bp was purified
from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). The fragment was then
30 cloned into a pCXND3 expression vector to construct pCXND3-VB22B sc(Fv)2.
[0186]
JP 2004-071763
2.4 Expression of single-chain anti-human Mpl antibody in animal cells
A cell line stably expressing the single-chain antibody was prepared from CHO-DG44
cells as described below. Gene transfer was achieved by electroporation using a Gene Pulser II
(Bio-Rad). An expression vector (25 fig) and 0.75 mL of CHO-DG44 cells suspended in PBS
5 (1 x 10 7 cells/mL) were mixed. The resulting mixture was cooled on ice for 10 min, transferred
into a cuvette, and pulsed at 1.5-kV and 25 p,FD. After a ten-minute restoration period at room
temperature, the electroporated cells were plated in CHO-S-SFMII medium (Invitrogen)
containing 500 jig/mL Geneticin (Invitrogen). CHO cell lines expressing the single-chain
antibody were established through selection. A cell line stably expressing VB22B sc(Fv)2 and
1 0 its culture supernatants were obtained by this method.
[0187]
The transient expression of the single-chain antibody was achieved using COS7 cells as
described below. An expression vector (10 jig) and 0.75 mL of COS7 cells suspended in PBS
(1x10' cells/mL) were mixed. The resulting mixture was cooled on ice for 10 min, transferred
1 5 into a cuvette, and then pulsed at 1 .5-kV and 25 \xFD. After a ten-minute restoration period at
room temperature, the electroporated cells were plated in DMEM/10% FBS medium (Invitrogen).
The cells were incubated overnight and then washed with PBS. CHO-S-SFMII medium was
added and the cells were cultured for about three days. The culture supernatants for preparing
the VB22B diabody were thus prepared.
20 [0188]
2.5 Quantitation of single-chain anti-human Mpl antibodies in culture supernatants
The culture supernatant concentration of the single-chain anti-human Mpl antibody
transiently expressed in COS cells was determined using surface plasmon resonance. A sensor
chip CM5 (Biacore) was placed in Biacore 2000 (Biacore). ANTI-FLAG® M2 Monoclonal
25 Antibody (Sigma- Aldrich) was immobilized onto the chip. An appropriate concentration of
sample was injected over the chip surface at a flow rate of 5 mL/sec, and 50 mM diethylamine
was used to dissociate the bound antibody. Changes in the mass during sample injection were
recorded, and the sample concentration was calculated from the calibration curve prepared using
the mass changes of a standard sample. dbl2E10 (see WO 02/33073 and WO 02/33072) was
30 used as the diabody standard, and 12E10 sc(Fv)2 which has the same gene structure as that of
sc(Fv) 2 was used as the sc(Fv)2 standard.
[0189]
2.6 Purification of anti-human Mpl diabodies and single-chain antibodies
The culture supernatants of VB22B diabody-expressing COS7 cells or CHO cells was
35 loaded onto an Anti-Flag M2 Affinity Gel (Sigma- Aldrich) column equilibrated with a 50 mM
Tris-HCl buffer (pH7.4) containing 150 mM NaCl and 0.05% Tween20. The absorbed
64 JP 2004-071763
antibodies were eluted with 100 mM glycine-HCl (pH3.5). The fractions eluted were
immediately neutralized with 1 M Tris-HCl (pH8.0), and loaded onto a HiLoad 26/60 Superdex
200 pg (Amersham Biosciences) column for gel filtration chromatography. PBS/0.01%
Tween20 was used in the gel filtration chromatography.
5 [0190]
VB22B sc(Fv) 2 was purified from the culture supernatants of VB22B sc(Fv)2-expressing
COS7 cells or CHO cells under the same conditions used for purifying the diabodies. A
large-scale preparation of VB22B sc(Fv)2 was prepared by loading the CHO cell culture
supernatants onto a Macro-Prep Ceramic Hydroxyapatite Type I (Bio-Rad) column equilibrated
10 with a 20 mM phosphate buffer (pH6.8), and eluting the VB22B sc(Fv) 2 in a stepwise manner
with 250 mM phosphate buffer (pH6.8). The eluted fraction was concentrated on an ultrafilter,
and then fractionated by gel filtration chromatography using a HiLoad 26/60 Superdex 200 pg
(Amersham Biosciences) column, and a fraction corresponding to the molecular weight range of
about 40 kD to 70 kD was obtained. The fraction was loaded onto an Anti-Flag M2 Affinity
15 Gel column equilibrated with a 50 mM Tris-HCl buffer (pH7.4) containing 150 mM NaCl and
0.05% Tween20. The absorbed antibody was eluted with 100 mM glycine-HCl (pH3.5). The
eluted fraction was immediately neutralized with 1 M Tris-HCl (pH8.0), and loaded onto a
HiLoad 26/60 Superdex 200 pg (Amersham Biosciences) column for gel filtration
chromatography. 20 mM acetate buffer (pH6.0) containing 150 mM NaCl and 0.01% Tween80
20 was used in the gel filtration chromatography. In each purification step, the presence of the
diabody and sc(Fv)2 in the samples was confirmed by SDS-PAGE and Western blotting using an
anti-Flag antibody (Sigma-Aldrich).
[0191]
2.7 Binding activity analyses of single-chain anti-human Mpl antibodies by flow cytometry
25 CHO-human Mpl, CHO-monkey Mpl, and CHO-mouse Mpl cells were recovered and
suspended in FACS buffer (1% FBS/PBS) to a final concentration of 1 x 10 6 cells/mL. Cell
suspensions were aliquoted at 100-jaL/well into the Multiscreen-HV Filter Plates (Millipore).
After centrifugation, the supernatant was removed. An appropriate concentration of diabody or
sc(Fv)2 was added into each well and incubated on ice for 30 min. The cells were washed once
30 with 200 |iL of FACS buffer, and incubated on ice for 30 min following the addition of 10 jag/mL
ANTI-FLAG® M2 Monoclonal Antibody (Sigma-Aldrich). The cells were then washed once
with 200 jj.L of FACS buffer, and a lOOx-diluted FITC-labeled anti-mouse IgG antibody
(Beckman Coulter) was added to the plate. The plate was incubated on ice for 30 min. After
centrifugation, the supernatant was removed. The cells were suspended in 400 jiL of FACS
35 Buffer, and then analyzed by flow cytometry using EPICS ELITE ESP (Beckman Coulter). An
analysis gate was set on the forward and side scatters of a histogram to include viable cell
65 JP 2004-071763
populations.
[0192]
The binding activity of the purified VB22B sc(Fv) 2 to various Mpl molecules expressed
in CHO cells was determined (Fig. 2). VB22B sc(Fv) 2 was found to specifically bind to
5 CHO-human Mpl and CHO-monkey Mpl but not to the host cell CHO or CHO-mouse Mpl.
This binding characteristic of VB22B sc(Fv)2 is comparable to those of VB22B IgG, indicating
that the antibody binding site remains unaltered by reduction of molecular weight.
[0193]
2.8 Analyses of TPO-like agonistic activity for single-chain anti-human Mpl antibodies
10 TPO-like agonistic activity was assessed using BaF3 -human Mpls or BaF3 -monkey
Mpls that proliferate in a TPO-dependent manner.
Cells from each cell line were washed twice with RPMI 1640/1% FBS (fetal bovine
serum) (Invitrogen), and then suspended in RPMI 1640/10% FBS to a concentration of 4 x 10 5
cells/mL. Cell suspensions were aliquoted at 60-jj.L/well into a 96-well plate. Various
1 5 concentrations of rhTPO (R&D) and COS7 culture supernatants or purified samples were
prepared, and a 40-(j,L aliquot was added into each well. The plates were then incubated at
37°C under 5% C0 2 for 24 hr. Immediately after a 10-p.L aliquot of WST-8 reagent (Cell
Count Reagent SF; Nacalai Tesque) was added into each well, absorbance was measured at 450
nm (and at 655 nm as a control) using Benchmark Plus. After two hours of incubation,
20 absorbance was again measured at 450 nm (and at 655 nm as a control). The WST-8 reagent
changes colors at 450 nm in a color reaction that reflects the viable cell count. The TPO-like
agonistic activity was assessed using the change in absorbance during the two-hour incubation as
an index. EC50 values were computed using GraphPad Prism.
[0194]
25 TPO-like agonistic activity was assayed using the human leukemia cell line M-07e
(purchased from DSMZ) which proliferates TPO-dependently. M-07e cells were washed twice
with RPMI 1640/1% FBS, and then suspended in RPMI 1640/10% FBS to a concentration of 5 x
10 5 cells/mL. The resulting cell suspension was aliquoted at 50-p.L/well into a 96-well plate.
Various concentrations of rhTPO and COS7 culture supernatants or purified samples were
30 prepared, and a 50-jj.L aliquot was added into each well. The plates were then incubated at
37°C under 5% C0 2 for 48 hr. Immediately after a 10-^L aliquot of WST-8 reagent (Cell
Count Reagent SF; Nacalai Tesque) was added to each well, absorbance of was measured at 450
nm (and at 655 nm as a control) using a Benchmark Plus. After four hours of incubation,
absorbance was again measured at 450 nm (and at 655 nm as a control). The TPO-like
35 agonistic activity was assayed using the change in absorbance during the four-hour incubation as
an index.
66 JP 2004-071763
[0195]
Purified VB22B IgG, VB22B diabody, and VB22B sc(Fv) 2 were assayed for their
TPO-like agonistic activities using BaF3 -human Mpl, BaF3 -monkey Mpl 5 and M-07e. The
results are shown in Figures 3, 4, and 5, respectively. The presence of bivalent antigen-binding
5 domains in a single antibody molecule is essential for its agonistic activity. The distance and
angle between two antigen-binding domains can also be important factors (see WO 02/33073
and WO 02/33072). Similar results were obtained for the newly isolated anti-human Mpl
antibodies. Specifically, the agonistic activities of VB22B diabody and VB22B sc(Fv) 2 (EC50 =
61 pM and 27 pM in BaF-human Mpl, respectively) were higher than that of VB22B IgG (EC50
10 > 30 nM in BaF-human Mpl), and were equivalent to or higher than that of the
naturally-occurring human TPO ligand (EC50 = 76 pM in BaF-human Mpl). The VB22B
diabody activity was lower than that of VB22B sc(Fv)2. This suggests that the structure of a
single-chain antibody is greatly altered by its molecular shape and the length of the linker
sequence, which in turn changes the agonistic activity. Sixteen types of the single-chain
1 5 anti-human Mpl antibodies were obtained, each exhibiting a high agonistic activity. The amino
acid sequences of the H chain and L chain variable regions of the representative antibodies are
shown in Figures 6 and 7, respectively.
[0196]
2.9 Humanization of single-chain anti-human Mpl antibody
20 Antibody sequence data for the humanization of VB22B sc(Fv)2 were obtained from the
Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/), and homology searches were carried
out independently for the H chain variable region and the L chain variable region. As a result,
the H chain variable region was found to be highly homologous to DN13 (Smithson S. L. et al. 9
Mol Immunol. (1999) 36: 113-124). The L chain variable region was found to be highly
25 homologous to ToP027 (Hougs L. et ai, J. Immunol. (1999) 162: 224-237). Humanized
antibodies were prepared by inserting a complementarity-determining region (hereinafter
abbreviated as "CDR") into the framework regions (hereinafter abbreviated as "FR") of the
above antibodies. The humanized antibody sc(Fv) 2 was expressed in CHO-DG44 cells, and its
agonistic activity was assessed using BaF-human Mpl. The agonistic activity was used as an
30 index to generate a humanized VB22B sc(Fv)2 which has agonistic activity equivalent to that of
murine VB22B sc(Fv)2 by replacing one or more amino acids in its framework region.
[0197]
Specifically, synthetic oligo-DNAs of approximately 50 nucleotides in length were
designed as to make 20 of these nucleotides available for hybridization, and the synthetic
35 oligo-DNAs were assembled by PCR to prepare genes that encode the respective variable
regions. Using the resulting genes, sc(Fv)2 was similarly prepared by the method described in
JP 2004-071763
Example 2.3. The respective DNAs were cloned into a pCXND3 expression vector to construct
three kinds of expression vectors, pCXND3-hVB22B p-z sc(Fv) 2 , pCXND3-hVB22B g-e sc(Fv) 2 ,
and pCXND3-hVB22B e sc(Fv) 2 , to which the humanized VB22B sc(Fv) 2 is inserted. The
nucleotide sequence and the amino acid sequence of hVB22B p-z sc(Fv) 2 in the plasmid are
5 shown in SEQ ID NOs: 1 and 2. The nucleotide sequence and the amino acid sequence of
hVB22B g-e sc(Fv) 2 are shown in SEQ ID NOs: 253 and 254. The nucleotide sequence and the
amino acid sequence of hVB22B e sc(Fv) 2 are shown in SEQ ID NOs: 259 and 260. The
nucleotide sequence and the amino acid sequence of murine VB22B sc(Fv) 2 are shown in SEQ
ID NOs: 263 and 264. The plasmids were expressed in CHO-DG44 cells by the method
10 described in Example 2.4 and the purification was done using the method described in Example
2.6. The TPO-like agonistic activities of the purified samples were similarly determined using
the method described in Example 2.8. The TPO-like agonistic activities of the purified murine
VB22B sc(Fv) 2 , and humanized hVB22B e sc(Fv) 2 and hVB22B g-e sc(Fv) 2 in BaF3 -human
Mpl were assessed. The results are shown in Figures 19. The humanized VB22B sc(Fv) 2
1 5 showed comparable agonistic activities, suggesting that the humanization has no influence on the
activity.
[0198]
[Example 3] Preparation of anti-Mpl diabodies by the AGS method
Anti-Mpl diabodies having agonistic activity were prepared by an Autocrine Growth
20 Selection (AGS) method (see, WO 03/91424).
[0199]
3 . 1 Construction of a retrovirus library
Spleens were isolated from MRL/lpr mice immunized with shMPL-Flag by the method
described in Example 1.5, and homogenized in TRIZOL Reagent (Invitrogen) using a Dounce
25 homogenizer. After chloroform addition, the homogenized sample was shaken vigorously, the
aqueous phase was removed and total RNA was extracted by isopropanol precipitation. mRNA
was purified using a PolyATract System 1000 (Promega). Reverse transcription of 2.5 |ug
mRNA was carried out at 42°C for 50 min using the Superscript First strand synthesis system for
RT-PCR (Invitrogen) and the included oligo-dT primers to prepare cDNA.
30 [0200]
The composition of the PCR reaction solution (250 |iL) is shown below.
1 Ox KOD Plus Buffer (Toyobo) 25
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (Toyobo) 25 nL
2.5 mM MgS0 4 (Toyobo) 10
KOD Plus (Toyobo) 7.5
Reverse transcription products 25 jiL
68 JP 2004-071763
Mixed primers complementary to H chain or L chain variable region 500 pmol
[0201]
The reaction conditions were:
98 °C (initial temperature) for 3 min;
32 cycles of 98°C for 20 sec, 58°C for 20 sec, and 72°C for 30 sec;
5 and final extension was at 72 °C for 6 min.
[0202]
The H chain primer mix contained HS1 to HS19 (SEQ ID NOs: 178 to 196) and HA1 to
HA4 (SEQ ID NOs: 197 to 200), which were mixed at the indicated ratios next to the sequence
names in Table 1. The L chain primer mix contained LSI to LSI 7 (SEQ ID NOs: 201 to 217),
10 LSlambda (SEQ ID NO: 218), LAI to LA5 (SEQ ID NOs: 219 to 222), and LAlambda (SEQ ID
NO: 223). The respective PCR products were purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN). The H chain and L chain variable regions were linked via the
(Gly 4 Ser)i linker sequence by PCR using sc-S (SEQ ID NO: 224) and sc-AS (SEQ ID NO: 225)
as described below.
15 [0203]
The composition of the PCR reaction solution (100 jaL in total) is shown below.
1 Ox KOD Plus Buffer (Toyobo) 1 0 jaL
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (Toyobo) 10 ^L
2.5 mM MgS0 4 (Toyobo) 4 p.L
KOD Plus (Toyobo) 2 yL
Fragment of H chain variable region 4 jiL
Fragment of L chain variable region 4 jaL
[0204]
The first-round PCR conditions were:
94°C (initial temperature) for 3 min; and
20 seven cycles of 94°C for 1 min and 63 °C for 4 min.
Then, sc-S and sc-AS (25 pmol each) were added to the first-round products.
The second-round PCR conditions were:
30 cycles of 94°C for 30 sec, 55°C for 2 min, and 72°C for 2 min;
and final extension was at 72°C for 6 min.
25 [0205]
The resulting product with an Sfil restriction site at both ends was purified using the
QIAquick PCR Purification Kit (QIAGEN), and incubated with the Sfil restriction enzyme
(TaKaRa) overnight at 50°C. The PCR product purified from agarose gel using the QIAquick
Gel Extraction Kit (QIAGEN) was inserted into the Sfil site of the viral vector
JP 2004-071763
pMX/IL3ssGFPHis.
[0206]
The resulting plasmid was constructed by inserting a GFP gene, which has an EcoRI site,
mouse IL-3 signal sequence and Sfil site at its 5' end; and an Sfil site, His tag sequence,
5 termination codon, and Notl site at its 3' end, between the EcoRI and Noil sites on the pMX viral
vector (Onishi, M. et aL, Mol. Cell. Biol. 18: 3871-3879). The plasmid was introduced into the
ElectroMAX DH10B Tl phage resistant cells (Invitrogen) by electroporation (settings: 2.5 kV,
25 |iF, and 100Q) using a Gene Pulser II (Bio-Rad). The cells were plated onto an LB- Agar
plate containing 100 ng/mL ampicillin. After overnight incubation, 1 x 10 7 colonies were
10 obtained. Colonies were recovered from the plate and plasmids were then extracted using the
QIAGEN Plasmid Maxi Kit (QIAGEN).
[0207]
70 JP 2004-071763
[Table 1]
SEQ ID NO: 178
SEQ ID NO: 1 79
SEQ ID NO: 180
SEQ ID NO: 1 81
SEQ ID NO: 1 82
SEQ ID NO: 183
SEQ ID NO: 1 84
SEQ ID NO: 185
SEQ ID NO: 186
SEQ ID NO: 187
| SEQ ID NO: 188
SEQ ID NO: 189
ISEQ ID NO: 1 90
SEQ ID NO: 191
SEQ ID NO: 1 92
SEQ ID NO: 193
IsEQ ID NO: 1 94
SEQ ID NO: 195
SEQ ID NO: 196
SEQ ID NO: 197
SEQ ID NO: 198
SEQ ID NO: 199
SEQ ID NO: 200
SEQ ID NO: 201
SEQ ID NO: 202
SEQ ID NO: 203
IsEQ ID NO: 204
SEQ ID NO: 205
SEQ ID NO: 206
SEQ ID NO: 207
|SEQ ID NO: 208
SEQ ID NO: 209
SEQ ID NO: 210
SEQ ID NO: 21 1
SEQ ID NO:212
SEQ ID NO: 21 3
SEQ ID NO: 21 4
I SEQ ID NO: 21 5
SEQ ID NO: 21 6
SEQ ID NO : 2 1 7
SEQ ID NO: 21 8
SEQ ID NO: 21 9
SEQ ID NO: 220
ISEQ ID NO: 221
SEQ ID NO: 222
SEQ ID NO: 223
(HS1 (4))
(HS2 (4))
(HS3 (3))
(HS4 (4) )
(HS5(7))
(HS6 (2) )
(HS7 (1))
(HS8 (2) )
(HS9(5))
(HS10 (2)
(HS11 (2)
(HS12 (2)
(HS13 (1)
(HS14 (2)
(HS15 (2)
(HS16 (5)
GCCCAGCCGGCCATGGCGGAKGTRMAGCTTCAGGAGTC
GCCCAGCCGGCCATGGCGGAGGTBCAGCTBCAGCAGTC
GCCCAGCCGGCCATGGCGCAGGTGCAGCTGAAGSASTC
GCCCAGCCGGCCATGGCGGAGGTCCARCTGCAACARTC
GCCCAGCCGGCCATGGCGCAGGTYCAGCTBCAGCARTC
GCCCAGCCGGCCATGGCGCAGGTYCARCTGCAGCAGTC
GCCCAGCCGGCCATGGCGCAGGTCCACGTGAAGCAGTC
GCCCAGCCGGCCATGGCGGAGGTGAASSTGGTGGAATC
GCCCAGCCGGCCATGGCGGAVGTGAWGYTGGTGGAGTC
GCCCAGCCGGCCATGGCGGAGGTGCAGSKGGTGGAGTC
GCCCAGCCGGCCATGGCGGAKGTGCAMCTGGTGGAGTC
GCCCAGCCGGCCATGGCGGAGGTGAAGCTGATGGARTC
GCCCAGCCGGCCATGGCGGAGGTGCARCTTGTTGAGTC
GCCCAGCCGGCCATGGCGGARGTRAAGCTTCTCGAGTC
GCCCAGCCGGCCATGGCGGAAGTGAARSTTGAGGAGTC
GCCCAGCCGGCCATGGCGCAGGTTACTCTRAAAGWGTSTG
(HS17 (3.5)) GCCCAGCCGGCCATGGCGCAGGTCCAACTVCAGCARCC
(HS 1 8 ( 0 . 7 ) ) GCCCAGCCGGCCATGGCGGATGTGAACTTGGAAGTGTC
(HS1 9 (0.7)) GCCCAGCCGGCCATGGCGGAGGTGAAGGTCATCGAGTC
(HA1 ( 1 ) ) GGAGCCGCCGCCGCCCGAGGAAACGGTGACCGTGGT
GGAGCCGCCGCCGCCCGAGGAGACTGTGAGAGTGGT
GGAGCCGCCGCCGCCCGCAGAGACAGTGACCAGAGT
GGAGCCGCCGCCGCCCGAGGAGACGGTGACTGAGGT
GGCGGCGGCGGCTCCGAYATCCAGCTGACTCAGCC
GGCGGCGGCGGCTCCGAYATTGTTCTCWCCCAGTC
GGCGGCGGCGGCTCCGAYATTGTGMTMACTCAGTC
(LS4 (3.5)) GGCGGCGGCGGCTCCGAYATTGTGYTRACACAGTC
(LS 5 ( 4 ) ) GGCGGCGGCGGCTCCGAYATTGTRATGACMCAGTC
(LS6 ( 7 ) ) GGCGGCGGCGGCTCCGAYATTMAGATRAMCCAGTC
(LS7 ( 6 ) ) GGCGGCGGCGGCTCCGAYATTCAGATGAYDCAGTC
(LS8 (1.5)) GGCGGCGGCGGCTCCGAYATYCAGATGACACAGAC
(LS9 (2 ) ) GGCGGCGGCGGCTCCGAYATTGTTCTCAWCCAGTC
(LSI 0 (3.5)) GGCGGCGGCGGCTCCGAYATTGWGCTSACCCAATC
(HA2 ( 1 ) )
(HA3 (1) )
(HA4 ( 1 ) )
(LSI (1) )
(LS2 (2))
(LS3 (5) )
(LS11 (8)
(LS12 (8)
(LS13 (6)
(LS14 (2)
(LS15 (2)
(LS16 (1)
(LS17 (1)
GGCGGCGGCGGCTCCGAYATTSTRATGACCCARTC
GGCGGCGGCGGCTCCGAYRTTKTGATGACCCARAC
GGCGGCGGCGGCTCCGAYATTGTGATGACBCAGKC
GGCGGCGGCGGCTCCGAYATTGTGATAACYCAGGA
GGCGGCGGCGGCTCCGAYATTGTGATGACCCAGWT
GGCGGCGGCGGCTCCGAYATTGTGATGACACAACC
GGCGGCGGCGGCTCCGAYATTTTGCTGACTCAGTC
(LSlambda ( 1 ) ) GGCGGCGGCGGCTCCGATGCTGTTGTGACTCAGGAATC
(LAI ( 4 ) ) GGAATTCGGCCCCCGAGGCCTTGATTTCCAGCTTGG
(LA2 (4 ) ) GGAATTCGGCCCCCGAGGCCTTTATTTCCAGCTTGG
( LA4 ( 4 ) ) GGAATTCGGCCCCCGAGGCCTTTATTTCC AACTTTG
(LA5 ( 4 ) ) GGAATTCGGCCCCCGAGGCCTTCAGCTCCAGCTTGG
(LAlambda ( 1 ) ) GGAATTCGGCCCCCGAGGCCCCTAGGACAGTCAGTTTGG
JP 2004-071763
[0208]
3.2 Establishment of autonomously replicating cell lines by the AGS method
The resulting library was transfected into a packaging cell, Pt-E 5 (Morita, S. et al 9 Gene
therapy 7: 1063-1066) using FuGENE 6 (Roche Diagnostics). Specifically, Pt-E was plated
5 onto 6-cm dishes and cultured in DMEM/10% FBS (Invitrogen). A mixture of FuGENE 6 and
the library was added to the plate the following day. The culture medium was exchanged the
next day, and the culture supernatant was collected 24 hours after that. 10 jag/mL polybrene
(Hexadimethrine Bromide; Sigma) and 2 ng/mL mIL-3 were added to the culture supernatant
containing recombinant virus particles. The viral solution was used to infect the BaF-monkey
10 Mpl target cells. The cells were washed with PBS the following day, and suspended in RPMI
1640/10% FBS without mIL-3. The suspension was plated onto a 96-well plate at a cell density
of 1,000 cells/well. Autonomously replicating cell lines (AB317 and AB324) were obtained
after seven days of incubation. Genomic DNAs were extracted from these cells using a
DNeasy Tissue Kit (QIAGEN), and the antibody genes were amplified by PCR.
15 [0209]
The composition of the PCR reaction solution (50 \xL in total) is shown below.
1 Ox LA Taq Buffer (TaKaRa) 5 ^iL
2 mM dNTPs (dATP, dGTP, dCTP, and dTTP) (TaKaRa) 5 ^iL
2.5 mM MgCl 4 (TaKaRa) 5
TaKaRa LA Taq (TaKaRa) 0.5 \xL
Genomic DNA 0.5 jag
AGSdbS 1 (SEQ ID NO: 226) and AGSdbAl (SEQ ID NO: 227) 25 pmol
[0210]
The reaction conditions were:
94°C (initial temperature) for 1 min;
20 30 cycles of 94°C for 30 sec, 60°C for 30 sec, and 70°C for 1 min;
and final extension was at 72°C for 6 min.
[0211]
The nucleotide sequence and the amino acid sequence of the H chain of cloned AB3 17
are shown in SEQ ID NOs: 154 and 155. The nucleotide sequence and the amino acid
25 sequence of AB3 17 L chain are shown in SEQ ID NOs: 156 and 157. The nucleotide sequence
and the amino acid sequence of AB324 H chain are shown in SEQ ID NOs: 158 and 1 59. The
nucleotide sequence and the amino acid sequence of AB324 L chain are shown in SEQ ID NOs:
160 and 161.
[0212]
30 3.3 Activity assays of the diabodies obtained by AGS method
72
JP 2004-071763
Each of the anti-Mpl diabodies obtained above was inserted into the pCXND3
expression vector. The PCR primers used are a synthetic oligonucleotide complementary to the
5 5 end of the diabody and containing an EcoRI site, and a synthetic oligonucleotide
complementary to the nucleotide sequence of the 3 5 end of the diabody and containing a FLAG
5 tag and a Noil site. The PCR product thus obtained was inserted into pCXND3 between the
EcoRI and Notl sites. The diabody was expressed transiently in COS7 cells by the method
described in Example 2.4. The culture supernatant was removed and the activity of the diabody
was evaluated.
[0213]
10 The binding activities of the diabodies were assessed by flow cytometry using CHO
cells that express Mpl derived from various species (Fig. 8). AB3 17 was proven to bind to
CHOmouse Mpl.
[0214]
The TPO-like agonistic activities of the diabodies were evaluated using BaF-human Mpl,
15 BaF-monkey Mpl, and BaF-mouse Mpl (Figures 9, 10, and 11). AB317 had the highest
agonistic activity against human, monkey, and mouse Mpl, whereas AB324 showed the highest
agonistic activity against human and monkey Mpl.
[0215]
This proves that anti-Mpl diabodies having high agonistic activity can be obtained by
20 the AGS method.
[0216]
[Example 4] Agonistic activity assays of the anti-Mpl antibodies against mutant Mpl in
congenital amegakaryocytic thrombocytopenia (CAMT) patients
4.1 Establishment of BaF3 cell lines introduced with the mutant Mpl observed in CAMT patients
25 Mutations on G305C (R102P), C769T (R257C), and C823A (P275T) have been
reported in the Mpl gene of CAMT patients. The respective expression vectors carrying the
Mpl gene mutations were constructed and introduced into BaF3 cells. The following Mpl gene
fragments were constructed: normal Mpl gene (nucleotide sequence, SEQ ID NO: 246; amino
acid sequence, SEQ ID NO: 123); gene G305C in which C is substituted for 305th nucleotide G
30 relative to the initiation codon (nucleotide sequence, SEQ ID NO: 247; amino acid sequence,
SEQ ID NO: 248); gene C769T in which T is substituted for 769th nucleotide C (nucleotide
sequence, SEQ ID NO: 249; amino acid sequence, SEQ ID NO: 250); and gene C823A in which
A is substituted for 823rd nucleotide C (nucleotide sequence, SEQ ID NO: 251; amino acid
sequence, SEQ ID NO: 252). These DNA fragments were digested with EcoRI and Sail, and
35 inserted between the EcoRI and Sail sites on the animal cell expression vector pCOS2-Ha to
prepare pCOS2-hMPLfullG305C, P COS2-hMPLfullC769T, and pCOS2-hMPLfullC823 A.
73
JP 2004-071763
[0217]
The genes were introduced into BaF3 cells by the procedure described in Example 1.1.1
to establish BaF3 cell lines expressing each Mpl gene: BaF3-human MPL (G305C),
BaF3-human MPL (C769T), and BaF3-human MPL (C823A). After the selection, the cells
5 were cultured and passaged using RPMI 1640 containing 1 ng/mL mIL-3 and 10% FBS.
[0218]
4.2 Preparation of anti-human Mpl diabody and sc(Fv)2
Among the amino acid sequences shown in Figures 6 and 7, expression vectors were
prepared for the diabodies VB8B, VB45B, VB33, VB140, VB157, and TA136 using the same
10 procedure described in Example 2.2.2. The prepared expression vectors were introduced into
COS7 cells by the same procedure described in Example 2.2.4. The supernatant concentration
of each diabody was determined by the method of Example 2.2.5. The sc(Fv)2 expression
vector for TA136 was prepared by the same procedure described in Example 2.2.3. The vector
was introduced into CHO-DG44 cells by the same procedure described in Example 2.2.4.
1 5 sc(Fv)2 was purified from the culture supernatant thus obtained using the same method described
in Example 2.2.6.
[0219]
4.3 Agonistic activity assays of sc(Fv)2 and the anti-human Mpl diabodies
The prepared diabodies and sc(Fv)2 were assayed for their agonistic activities in normal
20 Mpl and mutant Mpl in BaF3 cells by the same procedure described in Example 2.2.8. The
agonistic activities in BaF3 -human Mpl and BaF3-human Mpl (G305C) were compared using
the culture supernatants of cells expressing the diabodies. The TA136 diabody (TA136 db) was
shown to have a low agonistic activity in BaF3 -human Mpl cells expressing the normal Mpl gene,
and a high agonistic activity in BaF3 -human Mpl (G305C) cells expressing the mutant Mpl gene.
25 hTPO and the rest of the diabodies did not show a high agonistic activity in BaF3 -human Mpl
(G305C) cells (Figures 12 and 13).
[0220]
In addition, the agonistic activities of the TA136 diabody and TA136 sc(Fv)2 in
BaF3 -human Mpl, BaF3 -human Mpl (G305C), BaF3 -human Mpl (C769T), and BaF3 -human
30 Mpl (C823A) cells were assessed using a purified sample of the diabody. Compared with
hTPO and the TA136 diabody, TA136 sc(Fv)2 exhibited a higher agonistic activity in all three
types of the TPO receptor mutant cell lines (Figures 15, 16 and 17). Furthermore, it was shown
that in BaF3 -human Mpl cells expressing the normal Mpl gene, the TA136 diabody exhibited a
lower activity than hTPO. However, an agonistic activity equivalent to that of hTPO was
35 achieved by converting the diabody into sc(Fv)2 (Fig. 14).
74
JP 2004-071763
[Brief Description of the Drawings]
[0221]
[Fig. 1] Fig. 1 demonstrates the strategy for preparing single-chain antibody sc(Fv)2.
[Fig. 2] Fig. 2 illustrates the assessment of VB22B sc(Fv)2 binding activity using an
5 Mpl-expressing CHO cell line. Purified VB22B sc(Fv)2 was used.
[Fig. 3] Fig. 3 illustrates the assessment of VB22B antibody agonistic activity using
BaF-human Mpl.
[Fig. 4] Fig. 4 illustrates the assessment of VB22B antibody agonistic activity using
BaF-monkey Mpl.
10 [Fig. 5] Fig. 5 illustrates the assessment of VB22B antibody agonistic activity using
M-07e.
[Fig. 6] Fig. 6 shows the amino acid sequences of anti-human Mpl antibodies (H chains)
that exhibit higher agonistic activities when converted into minibodies.
[Fig. 7] Fig. 7 shows the amino acid sequences of anti-human Mpl antibodies (L chains)
1 5 which exhibit higher agonistic activities when converted into minibodies.
[Fig. 8] Fig. 8 illustrates the binding activity assessment of AB317 diabody using
Mpl-expressing CHO cells. Both VB22B diabody (solid line) and AB3 17 diabody (broken
line) were obtained from COS7 culture supernatants.
[Fig. 9] Fig. 9 illustrates the agnostic activity assessment of AB324 and AB317
20 diabodies using BaF-human Mpl.
[Fig. 10] Fig. 10 illustrates the agnostic activity assessment of AB324 and AB317
diabodies using BaF-monkey Mpl.
[Fig. 11] Fig. 11 illustrates the agnostic activity assessment of AB324 and AB317
diabodies using BaF-mouse Mpl.
25 [Fig. 12] Fig. 12 shows the agonistic activities of diabodies and hTPO in BaF3 -human
Mpl cells. The Y-axis shows OD at 450/655 nm, and the X-axis represents concentration.
[Fig. 13] Fig. 13 shows the agonistic activities of diabodies and hTPO in BaF3-human
Mpl (G305C) cells. The Y-axis shows OD at 450/655 nm, and the X-axis represents
concentration.
30 [Fig. 14] Fig. 14 shows the agonistic activities of TA136 db and TA136 sc(Fv)2 in
BaF3-human Mpl cells. The Y-axis shows OD at 450/655 nm and the X-axis represents
concentration.
[Fig. 15] Fig. 15 shows the agonistic activities of TA136 db and TA136 sc(Fv) 2 in
BaF3-human Mpl (G305C) cells. The Y-axis shows OD at 450/655 nm, and the X-axis
35 represents concentration.
[Fig. 16] Fig. 16 shows the agonistic activities of TA136 db and TA136 sc(Fv)2 in
75
JP 2004-071763
BaF3-human Mpl (C769T) cells. The Y-axis shows OD at 450/655 nm, and the X-axis
represents concentration.
[Fig. 17] Fig. 17 shows the agonistic activities of TA136 db and TA136 sc(Fv)2 in
BaF3 -human Mpl (C823A) cells. The Y-axis shows OD at 450/655 nm, and the X-axis
5 represents concentration.
[Fig. 18] Fig. 18 shows the positions of FRs and CDRs in humanized heavy chain
sequences (hVB22B p-z: VH), and humanized light chain sequences (hVB22B p-z: VL).
[Fig. 19] Fig. 19 shows the TPO-like agonistic activities of murine VB22B sc(Fv)2,
hVB22B e sc(Fv) 2 , and hVB22B g-e sc(Fv)2 in BaF3 -human Mpl. The Y-axis shows
10 absorbance ratio (450nm/655nm), and the X-axis represents concentration.
76
[Sequence Listing]
SEQUENCE LISTING
<110> CHUGAI SEIYAKU KABUSH I K I KAISHA
<120> anti-Nlpl antibody
<130> C1-A0320Y1
<150> JP 2003-415746
<151> 2003-12-12
<160> 285
<170> Patentln version 3. 1
<210> 1
<211> 1572
<212> DNA
<213> Homo sapiens
<400> 1
atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag
gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc
tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gcagaggcct
ggaaagggtc ttgagtggat gggacggatt tatcctggag atggagaaac tatctacaat
gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat
gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt
77 JP 2004-071763
ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctgca 480
ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 540
5
ctcctgcata gtaatggcaa cacttacttg tattggttcc agcagaagcc agggcagtct 600
ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 660
10 ggcagtggat caggcacagc ttttacactg aaaatcagca gagtggaggc tgaggatgtt 720
ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 780
ctggaaatca aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840
15
gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 900
tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gcagaggcct 960
20 ggaaagggtc ttgagtggat gggacggatt tatcctggag atggagaaac tatctacaat 1020
gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 1080
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 1140
25
gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 1200
ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctgca 1260
30 ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 1320
ctcctgcata gtaatggcaa cacttacttg tattggttcc agcagaagcc agggcagtct 1380
ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 1440
35
ggcagtggat caggcacagc ttttacactg aaaatcagca gagtggaggc tgaggatgtt 1500
78
JP 2004-071763
ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 1560
ctggaaatca aa 1572
5
<210> 2
<211> 524
<212> PRT
10 <213> Homo sapiens
<400> 2
Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly
1 5 10 15
15
Val Gin Ser Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
20 35 40 45
Thr Asn Ser Trp Met Asn Trp Val Arg Gin Arg Pro Gly Lys Gly Leu
50 55 60
25 Glu Trp Met Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr lie Tyr Asn
65 70 75 80
Gly Lys Phe Arg Val Arg Val Thr Me Thr Ala Asp Glu Ser Thr Ser
85 90 95
30
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
35 115 120 125
79
JP 2004-07 1763
Gin Gly Thr
130
Gly Gly Ser
5 145
Leu Ser Leu
10 Ser Ser Lys
Phe Gin Gin
195
15
Ser Asn Leu
210
Gly Thr Ala
20 225
Gly Val Tyr
25 Gin Gly Thr
Gly Ser Gly
275
30
Glu Val Lys
290
Gly Tyr Thr
35 305
Thr Val Thr Val
135
Gly Gly Gly Gly
150
Pro Val Thr Pro
165
Ser Leu Leu His
180
Lys Pro Gly Gin
Ala Ser Gly Val
215
Phe Thr Leu Lys
230
Tyr Cys Met Gin
245
Lys Leu Glu Me
260
Gly Gly Gly Ser
Lys Pro Gly Ala
295
Phe Thr Asn Ser
310
Ser Ser Gly Gly Gly
140
Ser Asp Me Val Met
155
Gly Glu Pro Ala Ser
170
Ser Asn Gly Asn Thr
185
Ser Pro Gin Leu Leu
200
Pro Asp Arg Phe Ser
220
I le Ser Arg Val Glu
235
His Me Glu Tyr Pro
250
Lys Gly Gly Gly Gly
265
Gin Val Gin Leu Val
280
Ser Val Lys Val Ser
300
Trp Met Asn Trp Val
315
Gly Ser Gly Gly
Thr Gin Ser Ala
160
1 1 e Ser Cys Arg
175
Tyr Leu Tyr Trp
190
I le Tyr Arg Met
205
Gly Ser Gly Ser
Ala Glu Asp Val
240
Phe Thr Phe Gly
255
Ser Gly Gly Gly
270
Gin Ser Gly Pro
285
Cys Lys Ala Ser
Arg Gin Arg Pro
320
80 JP 2004-071763
Gly Lys Gly Leu Glu Trp Met Gly Arg Me Tyr Pro Gly Asp Gly Glu
325 330 335
Thr lie Tyr Asn Gly Lys Phe Arg Val Arg Val Thr lie Thr Ala Asp
5 340 345 350
Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
355 360 365
10 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe
370 375 380
Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
385 390 395 400
15
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Me Val Met
405 41 0 41 5
Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser
20 420 425 430
Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr
435 440 445
25 Tyr Leu Tyr Trp Phe Gin Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu
450 455 460
lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
465 470 475 480
30
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Me Ser Arg Val Glu
485 490 495
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His Me Glu Tyr Pro
35 500 505 510
81
JP 2004-071763
Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys
515 520
5 <210> 3
<211> 5
<212> PRT
<213> Mus musculus
10 <400> 3
Ser Ser Trp Met Asn
1 5
15 <210> 4
<211> 17
<212> PRT
<213> Mus musculus
20 <400> 4
Arg Thr Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
25
<210> 5
<211> 13
30 <212> PRT
<213> Mus musculus
<400> 5
Gly Trp lie Leu Ala Asp Gly Gly Tyr Ser Phe Ala Tyr
35 1 5 10
82
JP 2004-071763
<210> 6
<211> 5
<212> PRT
5 <213> Mus musculus
<400> 6
Ser Ser Trp Met Asn
1 5
10
<210> 7
<211> 17
<212> PRT
15 <213> Mus musculus
<400> 7
Arg I le Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
20
Gly
25 <210> 8
<211> 9
<212> PRT
<213> Mus musculus
30 <400> 8
Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr
1 5
35 <210> 9
<211> 5
83
JP 2004-071763
<212> PRT
<213> Mus musculus
<400> 9
5 Ser Ser Trp Met Asn
1 5
<210> 10
10 <211> 17
<212> PRT
<213> Mus musculus
<400> 1 0
15 Arg He Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
20
<210> 11
<211> 9
<212> PRT
25 <213> Mus musculus
<400>. 11
Gly Phe Gly Asp Tyr Ser Phe Ala Tyr
1 5
30
<210> 12
<211> 5
<212> PRT
35 <213> Mus musculus
84 JP 2004-071763
<400> 12
Ser Ser Trp Met Asn
1 5
5
<210> 13
<211> 17
<212> PRT
<213> Mus musculus
10
<400> 13
Arg lie Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
15 Gly
<210> 14
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 14
25 Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr
1 5
<210> 15
30 <211> 5
<212> PRT
<213> Mus musculus
<400> 1 5
Arg Ser Trp Met Asn
1 5
85
JP 2004-071763
<210> 16
<211> 17
5 <212> PRT
<213> Mus musculus
<400> 16
Arg lie Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
10 1 5 10 15
<210> 17
<211> 9
<212> PRT
<213> Mus musculus
<400> 1 7
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
Gly
25
<210>
18
<211>
5
<212>
PRT
<213>
Mus musculus
30
<400>
18
Asn Ser Trp Met Asn
5
35
<210> 19
86
JP 2004-071763
<211> 17
<212> PRT
<213> Mus musculus
5 <400> 19
Arg He Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe Lys
15 10 15
Gly
10
<210> 20
<211> 9
15 <212> PRT
<213> Mus musculus
<400> 20
Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
20 1 5
<210> 21
<211> 5
25 <212> PRT
<213> Mus musculus
<400> 21
Asn Tyr Trp Val Asn
30 1 5
<210> 22
<211> 17
35 <212> PRT
<213> Mus musculus
87
JP 2004-071763
<400> 22
Arg lie His Pro Ser Asp Ser Glu Thr His Cys Asn Gin Lys Phe Lys
15 10 15
5
Arg
10 <210> 23
<211> 6
<212> PRT
<213> Mus musculus
15 <400> 23
Gly Gly Trp Phe Ala Tyr
1 5
20 <210> 24
<211> 5
<212> PRT
<213> Mus musculus
25 <400> 24
Ser Ser Trp Met Asn
1 5
30 <210> 25
<211> 17
<212> PRT
<213> Mus musculus
35 <400> 25
Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe Lys
88
JP 2004-071763
15
25
35
10 15
Gly
<210> 26
<211> 9
<212> PRT
10 <213> Mus musculus
<400> 26
Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
1 5
<210> 27
<211> 5
<212> PRT
20 <213> Mus musculus
<400> 27
Thr Ser Trp Met Asn
1 5
<210> 28
<211> 17
<212> PRT
30 <213> Mus musculus
<400> 28
Arg He Tyr Pro Gly Asp Gly Glu Ala Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
89
JP 2004-071763
<210> 29
<211> 9
<212> PRT
<213> Mus musculus
<400> 29
10 Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
1 5
<210> 30
15 <211> 5
<212> PRT
<213> Mus musculus
<400> 30
20 Ser Ser Trp Met Asn
1 5
<210> 31
25 <211> 17
<212> PRT
<213> Mus musculus
<400> 31
30 Arg lie Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
Gly
35
90
JP 2004-071763
<210> 32
<211> 9
<212> PRT
<213> Mus musculus
<400> 32
Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr
1 5
<210> 33
<211> 5
<212> PRT
<213> Mus musculus
<400> 33
Arg Ser Trp Met Asn
1 5
20
<210> 34
<211> 17
<212> PRT
<213> Mus musculus
25
<400> 34
Arg lie Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys
15 10 15
30 Gly
35
<210> 35
<211> 9
<212> PRT
91
JP 2004-071763
<213> Mus musculus
<400> 35
Gly Asp Gly Asp Tyr Ser Phe Ala Tyr
5 1 5
<210> 36
<211> 5
10 <212> PRT
<213> Mus musculus
<400> 36
Asn Ser Trp Met Asn
15 1 5
<210> 37
<211> 17
20 <212> PRT
<213> Mus musculus
<400> 37
Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn Gly Lys Phe Arg
25 1 5 10 15
Val
30
35
<210> 38
<211> 9
<212> PRT
<213> Mus musculus
<400> 38
92
JP 2004-071763
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
5 <210> 39
<211> 5
<212> PRT
<213> Mus musculus
10 <400> 39
Asp Tyr Trp Val Asn
1 5
15 <210> 40
<211> 17
<212> PRT
<213> Mus musculus
20 <400> 40
Arg He His Pro Tyr Asp Ser G I u Thr His Tyr Asn Gin Lys Phe Lys
15 10 15
Asn
25
<210> 41
<211> 6
30 <212> PRT
<213> Mus musculus
<400> 41
Gly Gly Trp Phe Ala Ser
35 1 5
93
JP 2004-071763
<210> 42
<211> 5
<212> PRT
5 <213> Mus musculus
<400> 42
Asp Tyr Trp Met Asn
1 5
10
<210> 43
<211> 17
<212> PRT
15 <213> Mus musculus
<400> 43
Arg Me His Pro Phe Asp Ser Glu Thr His Cys Ser Gin Lys Phe Lys
15 10 15
20
Asn
25 <210> 44
<211> 6
<212> PRT
<213> Mus musculus
30 <400> 44
Gly Gly Trp Phe Ala Tyr
1 5
35 <210> 45
<211> 5
94
JP 2004-071763
<212> PRT
<213> Mus musculus
<400> 45
5 Asn Ser Trp Met Asn
1 5
<210> 46
10 <211> 17
<212> PRT
<213> Mus musculus
<400> 46
15 Arg lie Tyr Pro Gly Asp Gly Glu Thr He Tyr Asn Gly Lys Phe Arg
15 10 15
Val
20
<210> 47
<211> 9
<212> PRT
25 <213> Mus musculus
<400> 47
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
30
<210> 48
<211> 5
<212> PRT
35 <213> Mus musculus
95
JP 2004-071763
10
<400> 48
Asn Ser Trp Met Asn
1 5
<210> 49
<211> 17
<212> PRT
<213> Mus musculus
<400> 49
Arg Me Tyr Pro Gly Asp Gly Asp Thr He Tyr Asn Gly Asn Phe Lys
15 10 15
15 Gly
<210> 50
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 50
25 Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
<210> 51
30 <211> 5
<212> PRT
<213> Mus musculus
<400> 51
35 Ser Tyr Thr Met Ser
1 5
96
JP 2004-071763
<210> 52
<211> 17
5 <212> PRT
<213> Mus musculus
<400> 52
Thr Me Ser Ser Gly Ser Ser Thr lie Tyr Tyr Ala Asp Thr Val Lys
10 1 5 10 15
Gly
15
<210> 53
<211> 6
<212> PRT
<213> Mus musculus
20
<400> 53
Arg Trp Phe Leu Asp Cys
1 5
25
<210> 54
<211> 5
<212> PRT
<213> Mus musculus
30
<400> 54
Ser Ser Trp Met Asn
1 5
35
<210> 55
97
JP 2004-071763
<211> 17
<212> PRT
<213> Mus musculus
5 <400> 55
Arg lie Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
10
<210> 56
<211> 9
15 <212> PRT
<213> Mus musculus
<400> 56
Ala Arg Lys Thr Ser Trp Phe Ala Tyr
20 1 5
<210> 57
<211> 6
25 <212> PRT
<213> Mus musculus
<400> 57
Ser Asp Tyr Ala Trp Ser
30 1 5
<210> 58
<211> 16
35 <212> PRT
<213> Mus musculus
98
JP 2004-071763
<400> 58
Tyr lie Thr Tyr Ser G ly Tyr Ser lie Tyr Asn Pro Ser Leu Lys Ser
15 10 15
<210> 59
<211> 7
<212> PRT
10 <213> Mus musculus
15
25
<400> 59
Gly Tyr Asp Asn Met Asp Tyr
1 5
<210> 60
<211> 16
<212> PRT
20 <213> Mus musculus
<400> 60
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 61
<211> 7
<212> PRT
30 <213> Mus musculus
<400> 61
Arg Met Ser Asn Leu Ala Ser
99
JP 2004-071763
<210> 62
<211> 9
<212> PRT
<213> Mus musculus
5
<400> 62
Met Gin His Leu Glu Tyr Pro Phe Thr
1 5
10
<210> 63
<211> 16
<212> PRT
<213> Mus musculus
15
<400> 63
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
20
<210> 64
<211> 7
<212> PRT
<213> Mus musculus
25
<400> 64
Arg Met Ser Asn Leu Ala Ser
1 5
30
<210> 65
<211> 9
<212> PRT
<213> Mus musculus
35
<400> 65
100 JP 2004-071763
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
5 <210> 66
<211> 16
<212> PRT
<213> Mus musculus
10 <400> 66
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
15 <210> 67
<211> 7
<212> PRT
<213> Mus musculus
20 <400> 67
Arg Met Ser Asn Leu Ala Ser
1 5
25 <210> 68
<211> 9
<212> PRT
<213> Mus musculus
30 <400> 68
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
35 <210> 69
<211> 16
101 JP 2004-071763
<212> PRT
<213> Mus musculus
<400> 69
5 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 70
10 <211> 7
<212> PRT
<213> Mus musculus
<400> 70
15 Arg Met Ser Asn Leu Ala Ser
1 5
<210> 71
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 71
25 Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 72
30 <211> 16
<212> PRT
<213> Mus musculus
<400> 72
35 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
102
JP 2004-071763
<210> 73
<211> 7
5 <212> PRT
<213> Mus musculus
<400> 73
Arg Met Ser Asn Leu Ala Ser
10 1 5
<210> 74
<211> 9
15 <212> PRT
<213> Mus musculus
<400> 74
Met Gin His Leu Glu Tyr Pro Tyr Thr
20 1 5
<210> 75
<211> 16
25 <212> PRT
<213> Mus musculus
<400> 75
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
30 1 5 10 15
<210> 76
<211> 7
35 <212> PRT
<213> Mus musculus
103
JP 2004-071763
<400> 76
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 77
<211> 9
<212> PRT
10 <213> Mus musculus
<400> 77
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 78
<211> 16
<212> PRT
20 <213> Mus musculus
<400> 78
Arg Ser Ser Lys Ser Leu Leu Tyr Ser Asn Gly Asn lie Tyr Leu Tyr
15 10 15
<210> 79
<211> 7
<212> PRT
30 <213> Mus musculus
<400> 79
Arg Met Ser Asn Leu Ala Ser
1 5
35
104
JP 2004-071763
<210> 80
<211> 9
<212> PRT
<213> Mus musculus
<400> 80
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
10
15
20
25
<210> 81
<211> 16
<212> PRT
<213> Mus musculus
<400> 81
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 82
<211> 7
<212> PRT
<213> Mus musculus
<400> 82
Arg Met Ser Asn Leu Ala Ser
1 5
30
35
<210> 83
<211> 9
<212> PRT
<213> Mus musculus
<400> 83
105 JP 2004-071763
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 84
<211> 16
<212> PRT
<213> Mus musculus
10 <400> 84
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
15 <210> 85
<211> 7
<212> PRT
<213> Mus musculus
20 <400> 85
Arg Met Ser Asn Leu Ala Ser
1 5
25 <210> 86
<211> 9
<212> PRT
<213> Mus musculus
30 <400> 86
Met Gin His Val Glu Tyr Pro Tyr Thr
1 5
35 <210> 87
<211> 16
106
JP 2004-071763
<212> PRT
<213> Mus musculus
<400> 87
5 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 88
10 <211> 7
<212> PRT
<213> Mus musculus
<400> 88
15 Arg Met Ser Asn Leu Ala Ser
1 5
<210> 89
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 89
25 Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 90
30 <211> 16
<212> PRT
<213> Mus musculus
<400> 90
35 Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
107
JP 2004-071763
<210> 91
<211> 7
5 <212> PRT
<213> Mus musculus
<400> 91
Arg Met Ser Asn Leu Ala Ser
10 1 5
<210> 92
<211> 9
15 <212> PRT
<213> Mus musculus
<400> 92
Met Gin His Leu Glu Tyr Pro Tyr Thr
20 1 5
<210> 93
<211> 16
25 <212> PRT
<213> Mus musculus
<400> 93
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
30 1 5 10 15
<210> 94
<211> 7
35 <212> PRT
<213> Mus musculus
108
JP 2004-071763
<400> 94
Arg Met Ser Asn Leu Ala Ser
1 5
<210> 95
<211> 9
<212> PRT
10 <213> Mus musculus
<400> 95
Met Gin His Me Glu Tyr Pro Phe Thr
1 5
<210> 96
<211> 16
<212> PRT
20 <213> Mus musculus
<400> 96
Arg Ser Ser Lys Ser Leu Leu Tyr Ser Asn Gly Asn Thr Tyr Leu Tyr
1 5 10 15
<210> 97
<211> 7
<212> PRT
30 <213> Mus musculus
<400> 97
Arg Met Ser Asn Leu Ala Ser
1 5
35
109
JP 2004-071763
<210> 98
<211> 9
<212> PRT
<213> Mus musculus
<400> 98
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
10
<210> 99
<211> 16
<212> PRT
<213> Mus musculus
15
<400> 99
Arg Ser Ser Lys Ser Leu Leu Tyr Ser Asn Gly Asn Me Tyr Leu Tyr
15 10 15
20
<210> 100
<211> 7
<212> PRT
<213> Mus musculus
25
<400> 100
Arg Met Ser Asn Leu Ala Ser
1 5
30
35
<210> 101
<211> 9
<212> PRT
<213> Mus musculus
<400> 101
110
JP 2004-071763
Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 102
<211> 16
<212> PRT
<213> Mus musculus
10 <400> 102
Arg Ser Ser Lys Ser Leu Leu His Asn Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
15 <210> 103
<211> 7
<212> PRT
<213> Mus musculus
20 <400> 103
Arg Met Ser Asn Leu Ala Ser
1 5
25 <210> 104
<211> 9
<212> PRT
<213> Mus musculus
30 <400> 104
Met Gin His lie Glu Tyr Pro Phe Thr
1 5
35 <210> 105
<211> 16
Ill JP 2004-071763
<212> PRT
<213> Mus musculus
<400> 105
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 106
10 <211> 7
<212> PRT
<213> Mus musculus
<400> 1 06
15 Arg Met Ser Asn Leu Ala Ser
1 5
<210> 107
20 <211> 9
<212> PRT
<213> Mus musculus
<400> 107
25 Met Gin His Leu Glu Tyr Pro Tyr Thr
1 5
<210> 108
30 <211> 15
<212> PRT
<213> Mus musculus
35
<400> 1 08
Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gin
15 10 15
112
JP 2004-071763
<210> 109
<211> 7
5 <212> PRT
<213> Mus musculus
<400> 1 09
Gly Ala Ser Asn Val Glu Ser
10 1 5
<210> 110
<211> 9
15 <212> PRT
<213> Mus musculus
<400> 110
Gin Gin Ser Arg Lys Val Pro Trp Thr
20 1 5
<210> 111
<211> 11
25 <212> PRT
<213> Mus musculus
<400> 111
Lys Ala Ser Gin Asn Val Gly Asn Me He Ala
30 1 5 10
<210> 112
<211> 7
35 <212> PRT
<213> Mus musculus
113
JP 2004-071763
<400> 112
Leu Ala Ser Tyr Arg Tyr Ser
1 5
<210> 113
<211> 9
<212> PRT
10 <213> Mus musculus
<400> 113
Gin Gin Tyr Ser Ser Ser Pro Leu Thr
1 5
<210> 114
<211> 12
<212> PRT
20 <213> Mus musculus
<400> 114
Ser Ala Ser Ser Ser Val Ser Ser Ser His Leu Tyr
1 5 10
<210> 115
<211> 7
<212> PRT
30 <213> Mus musculus
<400> 115
Ser Thr Ser Asn Leu Ala Ser
1 5
114 JP 2004-071763
10
15
20
25
<210> 116
<211> 9
<212> PRT
<213> Mus musculus
<400> 1 1 6
His Gin Trp Ser Ser Tyr Pro Trp Thr
1 5
<210> 117
<211> 411
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (1)..(411)
<223>
<400> 117
atg gaa tgg cct ttg ate ttt etc ttc etc ctg tea gga act gca ggt 48
Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 10 15
gtc cac tec cag gtt cag ctg cag cag tct gga cct gag ctg gtg aag 96
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
30 cct ggg gee tea gtg aag att tec tgc aag get tct ggc tat gca ttc 144
Pro Gly Ala Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
35
act
Thr
aac tec tgg atg aac tgg gtg aag cag agg cct gga aag ggt ctt
Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu
50 55 60
192
115 JP 2004-071763
gag tgg att gga egg att tat cct gga gat gga gaa act ate tac aat 240
Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn
65 70 75 80
ggg aaa ttc agg gtc aag gee aca ctg act gca gac aaa tec tec age 288
Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
10 aca gee tac atg gat ate age age ctg aca tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp I le Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
tac ttc tgt gca aga ggc tat gat gat tac teg ttt get tac tgg ggc 384
15 Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
caa ggg act ctg gtc act gtc tct gca 411
Gin Gly Thr Leu Val Thr Val Ser Ala
20 130 135
<210> 118
<211> 137
25 <212> PRT
<213> Mus musculus
<400> 1 1 8
Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
30 1 5 10 15
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
35 Pro Gly Ala Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
116 JP 2004-071763
Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu
50 55 60
5 Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn
65 70 75 80
Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
10
Thr Ala Tyr Met Asp Me Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
15 115 120 125
Gin Gly Thr Leu Val Thr Val Ser Ala
130 135
20
<210> 119
<211> 396
<212> DNA
<213> Mus musculus
25
<220>
<221> CDS
<222> (1). . (396)
<223>
30
<400> 1 1 9
atg agg tgc eta get gag ttc ctg ggg ctg ctt gtg ttc tgg att cct 48
Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Phe Trp Me Pro
1.5 10 15
35
gga gec att ggg gat att gtg atg act cag get gca ccc tct ata cct 96
117 JP 2004-071763
Gly Ala I le Gly Asp I le Val Met Thr Gin Ala Ala Pro Ser I le Pro
20 25 30
gtc act cct gga gag tea gta tec ate tec tgt agg tct agt aag agt
5 Val Thr Pro Gly Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser
35 40 45
144
etc ctg cat agt aat ggc aac act tac ttg tat tgg ttc ctg cag agg
Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg
10 50 55 60
192
15
cca ggc cag tct cct caa etc ctg ata tat egg atg tec aac ctt gee
Pro Gly Gin Ser Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala
65 70 75 80
tea gga gtc cca gat agg ttc agt ggc agt ggg tea gga act get ttc
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
85 90 95
240
288
20 aca ctg aga ate agt aga gtg gag get gag gat gtg ggt gtt tat tac
Thr Leu Arg Me Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
100 105 110
336
tgt atg caa cat ata gaa tat cct ttt acg ttc gga teg ggg acc aag
25 Cys Met Gin His Me Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys
115 120 125
384
ctg gaa ata aaa
Leu Glu Me Lys
30 130
396
<210> 120
<211> 132
35 <212> PRT
<213> Mus musculus
118 JP 2004-071763
<400> 1 20
Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Phe Trp I le Pro
15 10 15
5
Gly Ala lie Gly Asp Me Val Met Thr Gin Ala Ala Pro Ser Me Pro
20 25 30
Val Thr Pro Gly Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser
10 35 40 45
Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg
50 55 60
15 Pro Gly Gin Ser Pro Gin Leu Leu I le Tyr Arg Met Ser Asn Leu Ala
65 70 75 80
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
85 90 95
20
Thr Leu Arg Me Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
100 105 110
Cys Met Gin His Me Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys
25 115 120 125
Leu Glu lie Lys
130
30
<210> 121
<211> 762
<212> DNA
<213> Mus musculus
35
<400> 121
119 JP 2004-071763
atggaatggc ctttgatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactcccag 60
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 120
5 tgcaaggctt ctggctatgc attcactaac tcctggatga actgggtgaa gcagaggcct 180
ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240
10
gggaaattca gggtcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300
gatatcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aggctatgat 360
gattactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc aggtggtggt 420
15 ggttcggata ttgtgatgac tcaggctgca ccctctatac ctgtcactcc tggagagtca 480
gtatccatct cctgtaggtc tagtaagagt ctcctgcata gtaatggcaa cacttacttg 540
20
tattggttcc tgcagaggcc aggccagtct cctcaactcc tgatatatcg gatgtccaac 600
cttgcctcag gagtcccaga taggttcagt ggcagtgggt caggaactgc tttcacactg 660
agaatcagta gagtggaggc tgaggatgtg ggtgtttatt actgtatgca acatatagaa 720
25 tatcctttta cgttcggatc ggggaccaag ctggaaataa aa 762
<210> 122
<211> 254
30 <212> PRT
<213> Mus musculus
<400> 122
Met Glu Trp Pro Leu lie Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
35 1 5 10 15
120 JP 2004-07 1763
Val His Ser
Pro Gly Ala
5 35
Thr Asn Ser
50
10 Glu Trp I le
65
Gly Lys Phe
15
Thr Ala Tyr
Tyr Phe Cys
20 115
Gin Gly Thr
130
25 Val Met Thr
145
Val Ser Me
30
Asn Thr Tyr
Gin Val Gin Leu
20
Ser Val Lys Me
Trp Met Asn Trp
55
Gly Arg Me Tyr
70
Arg Val Lys Ala
85
Met Asp I le Ser
100
Ala Arg Gly Tyr
Leu Val Thr Val
135
Gin Ala Ala Pro
150
Ser Cys Arg Ser
165
Leu Tyr Trp Phe
180
Gin Gin Ser Gly Pro
25
Ser Cys Lys Ala Ser
40
Val Lys Gin Arg Pro
60
Pro Gly Asp Gly Glu
75
Thr Leu Thr Ala Asp
90
Ser Leu Thr Ser Glu
105
Asp Asp Tyr Ser Phe
120
Ser Ala Gly Gly Gly
140
Ser Me Pro Val Thr
155
Ser Lys Ser Leu Leu
170
Leu Gin Arg Pro Gly
185
Glu Leu Val Lys
30
Gly Tyr Ala Phe
45
Gly Lys Gly Leu
Thr I le Tyr Asn
80
Lys Ser Ser Ser
95
Asp Ser Ala Val
110
Ala Tyr Trp Gly
125
Gly Ser Asp I le
Pro Gly Glu Ser
160
His Ser Asn Gly
175
Gin Ser Pro Gin
190
Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg
35 195 200 205
121 JP 2004-071763
Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me Ser Arg
210 215 220
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His Me Glu
5 225 230 235 240
Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
245 250
10
<210> 123
<211> 635
<212> PRT
<213> Homo sapiens
15
<400> 123
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
15 10 15
20 Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 25 30
Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
35 40 45
25
Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin
50 55 60
Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys Pro Leu Ser
30 65 70 75 80
Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro
85 90 95
35 Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys
100 105 110
122 JP 2004-071763
Asn Val Phe
115
5 Ser Val Gly
130
Ser Gin Pro
145
10
Me Ser Asp
Lys Asn Ser
15
Cys Cys Pro
195
20 Ser Pro Cys
210
Thr Ser Pro
225
25
Cys Leu 1 1 e
Arg Ser Glu
30
Ser Leu Pro
275
35 Leu Gin Cys
290
Leu Asn Gin Thr
Leu Pro Ala Pro
135
Gly Glu Leu Gin
150
Phe Leu Arg Tyr
165
Thr Gly Pro Thr
180
Ala Leu Gin Arg
Ala Gin Pro Thr
215
Ser Arg Glu Ala
230
Ser Gly Leu Gin
245
Pro Asp Gly I le
260
Val Thr Val Asp
Phe Thr Leu Asp
295
Arg Thr Gin Arg Val
120
Pro Ser lie lie Lys
140
lie Ser Trp Glu Glu
155
Glu Leu Arg Tyr Gly
170
Val lie Gin Leu Me
185
Pro His Ser Ala Ser
200
Met Pro Trp Gin Asp
220
Ser Ala Leu Thr Ala
235
Pro Gly Asn Ser Tyr
250
Ser Leu Gly Gly Ser
265
Leu Pro Gly Asp Ala
280
Leu Lys Asn Val Thr
300
Leu Phe Val Asp
125
Ala Met Gly Gly
Pro Ala Pro Glu
160
Pro Arg Asp Pro
175
Ala Thr Glu Thr
190
Ala Leu Asp Gin
205
Gly Pro Lys Gin
Glu Gly Gly Ser
240
Trp Leu Gin Leu
255
Trp Gly Ser Trp
270
Val Ala Leu Gly
285
Cys Gin Trp Gin
123 JP 2004-071763
Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala
305 310 315 320
5 Arg Cys Cys Pro Arg Asp Arg Tyr Pro Me Trp Glu Asn Cys Glu Glu
325 330 335
Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys
340 345 350
10
His Phe Lys Ser Arg Asn Asp Ser lie Me His Me Leu Val Glu Val
355 360 365
Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser Pro Phe Trp
15 370 375 380
Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu
385 390 395 400
20 Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp
405 410 415
Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His
420 425 430
25
Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr
435 440 445
Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg
30 450 455 460
Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro
465 470 475 480
35 Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser Leu Val Thr
485 490 495
124 JP 2004-071763
Ala Leu His Leu Vai Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu
500 505 510
5 Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
515 520 525
Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg
530 535 540
10
Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys
545 550 555 560
Glu Glu Val Glu Pro Ser Leu Leu Glu Me Leu Pro Lys Ser Ser Glu
15 565 570 575
Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met Asp Tyr Arg
580 585 590
20 Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro
595 600 605
Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me Ala Asn His
610 615 620
25
Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
30 <210> 124
<211> 122
<212> PRT
<213> Mus musculus
35 <400> 124
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
125 JP 2004-071763
10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
Gly Arg Thr Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
10 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
15 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Trp Me Leu Ala Asp Gly Gly Tyr Ser Phe Ala Tyr Trp
100 105 110
20
Gly Gin Gly Thr Leu Val Thr Val Ser Ala
115 120
25 <210> 125
<211> 112
<212> PRT
<213> Mus musculus
30 <400> 125
Asp Me Val Met Thr Gin Ala Ala Pro Ser Me Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
35 20 25 30
126 JP 2004-071763
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
5 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg He
65 70 75 80
10 Ser Arg Val Glu Ala Glu Asp Val Gly Me Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Phe Thr Phe Gly Thr Gly Thr Lys Leu Glu He Lys
100 105 110
15
<210> 126
<211> 118
<212> PRT
20 <213> Mus musculus
<400> 126
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
25
Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp He
30 35 40 45
Gly Arg He Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
35
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
127 JP 2004-071763
Me Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
5 Ala Arg Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
10
<210> 127
<211> 112
<212> PRT
15 <213> Mus musculus
<400> 127
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
20
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
25 35 40 45
Pro Gin Leu Leu I le Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
30 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
35
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
128 JP 2004-071763
100 105 110
<210> 128
5 <211> 118
<212> PRT
<213> Mus musculus
<400> 1 28
10 Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
15
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp lie
35 40 45
Gly Arg I le Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe
20 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
25 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Phe Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
30
Leu Val Thr Val Ser Ala
115
35 <210> 129
<211> 112
129 JP 2004-071763
<212> PRT
<213> Mus musculus
<400> 1 29
5 Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
10
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Ala Pro
15 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
20 Ser Arg Val Glu Thr Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
25
<210> 130
<211> 118
<212> PRT
30 <213> Mus musculus
<400> 1 30
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
35
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser
130 JP 2004-071763
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
Gly Arg lie Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
10 65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
15 Ala Ser Gly Tyr Ala Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
20
<210> 131
<211> 112
<212> PRT
25 <213> Mus musculus
<400> 131
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
30
Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 35 40 45
131 JP 2004-071763
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg I le
5 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
10 Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
100 105 110
<210> 132
15 <211> 118
<212> PRT
<213> Mus musculus
<400> 1 32
20 Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser
20 25 30
25
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp lie
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
30 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
35 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
132 JP 2004-071763
Ala Ser Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
5 Leu Val Thr Val Ser Ala
115
<210> 133
10 <211> 112
<212> PRT
<213> Mus musculus
<400> 1 33
15 Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
20
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
25 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
30 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
35
133 JP 2004-071763
<210> 134
<211> 118
<212> PRT
<213> Mus musculus
<400> 1 34
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
10 Ser Val Lys Me Ser Cys Arg Ala Phe Gly Tyr Ala Phe Ser Asn Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
15
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
20 65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
25 Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
30
<210> 135
<211> 112
<212> PRT
35 <213> Mus musculus
134 JP 2004-071763
<400> 135
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
5 Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
10
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Ala Phe Thr Leu Arg Me
15 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
20 Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
<210> 136
25 <211> 115
<212> PRT
<213> Mus musculus
<400> 136
30 Gin Val Gin Leu Gin Gin Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
35
Trp Val Asn Trp Val Lys Gin Arg Pro Gly Arg Gly Leu Glu Trp Me
135 JP 2004-071763
35 40 45
Gly Arg He His Pro Ser Asp Ser Glu Thr His Cys Asn Gin Lys Phe
50 55 60
5
Lys Arg Lys Ala Thr Leu Thr Val Asn Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Me Gin Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
10 85 90 95
Thr Ser Gly Gly Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr
100 105 110
15 Val Ser Ala
115
<210> 137
20 <211> 112
<212> PRT
<213> Mus musculus
<400> 1 37
25 Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser
20 25 30
30
Asn Gly Asn Me Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu
35 50
Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
55 60
136 JP 2004-071763
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
5 85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
100 105 110
10
<210> 138
<211> 118
<212> PRT
<213> Mus musculus
15
<400> 1 38
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
20 Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
25
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Asn Asn Gly Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
30 65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
35 Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
137 JP 2004-071763
Leu Val Thr Val Ser Ala
115
5
<210> 139
<211> 112
<212> PRT
<213> Mus musculus
10
<400> 139
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
15 Glu Ser Val Ser He Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
20
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Ala Phe Thr Leu Arg He
25 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
30 Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
100 105 110
<210>
35 <211>
<212>
140
118
PRT
138 JP 2004-071763
<213> Mus musculus
<400> 140
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
5 1 5 10 15
Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Thr Ser
20 25 30
10 Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Glu Ala Asn Tyr Asn Gly Lys Phe
50 55 60
15
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Ser Ala Tyr
65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
20 85 90 95
Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
25 Leu Val Thr Val Ser Ala
115
<210> 141
30 <211> 112
<212> PRT
<213> Mus musculus
<400> 141
35 Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
139 JP 2004-071763
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
5 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Met Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
10
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
15 85 90 95
Val Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
20
<210> 142
<211> 118
<212> PRT
<213> Mus musculus
25
<400> 1 42
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
30 Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30
Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Pro Glu Trp Me
35 40 45
35
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe
140 JP 2004-071763
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
5
Met 61 n Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Tyr Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
10 100 105 110
Leu Val Thr Val Ser Ala
115
15
<210> 143
<211> 112
<212> PRT
<213> Mus musculus
20
<400> 143
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
25 Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
30
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
35 65 70 75 80
141 JP 2004-07 1763
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys
5 100 105 110
<210> 144
<211> 118
10 <212> PRT
<213> Mus musculus
<400> 144
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Leu Asn Pro Gly Ala
15 1 5 10 15
Ser Val Lys I le Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Arg Ser
20 25 30
20 Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu Glu Trp I le
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe
50 55 60
25
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Gin Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
30 85 90 95
Ala Arg Gly Asp Gly Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
35 Leu Val Thr Val Ser Ala
115
142 JP 2004-071763
<210> 145
<211> 112
5 <212> PRT
<213> Mus musculus
<400> 1 45
Asp lie Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
10 1 5 10 15
Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
15 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
20
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
25 85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys
100 105 110
30
<210> 146
<211> 115
<212> PRT
<213> Mus musculus
35
<400> 1 46
143 JP 2004-071763
Gin Val Gin Leu Gin Gin Pro Gly Thr Glu Leu Val Arg Pro Gly Ala
15 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
5 20 25 30
Trp Val Asn Trp Val Lys Gin Arg Pro Gly Arg Gly Leu Glu Trp lie
35 40 45
10 Gly Arg lie His Pro Tyr Asp Ser Glu Thr His Tyr Asn Gin Lys Phe
50 55 60
Lys Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
15
I le Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Gly Gly Trp Phe Ala Ser Trp Gly Gin Gly Thr Leu Val Thr
20 100 105 110
Val Ser Ala
115
25
<210> 147
<211> 112
<212> PRT
<213> Mus musculus
30
<400> 1 47
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
35 Glu Ser Val Ser He Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser
20 25 30
144 JP 2004-071763
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
5 Pro Gin Leu Leu I le Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Thr Me
65 70 75 80
10
Ser Ser Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys
15 100 105 110
<210> 148
<211> 115
20 <212> PRT
<213> Mus musculus
<400> 148
Gin Val Gin Leu Gin
25 1 5
Ser Val Lys Leu Ser
20
30 Trp Met Asn Trp Val
35
Gly Arg I le His Pro
50
35
Lys Asn Lys Ala Thr
Gin Pro Gly Ala Glu
10
Cys Lys Ala Ser Gly
25
Lys Gin Arg Pro Gly
40
Phe Asp Ser Glu Thr
55
Leu Thr Val Asp Lys
Leu Val Lys Pro Gly Ala
15
Tyr Thr Phe Thr Asp Tyr
30
Arg Gly Leu Glu Trp I le
45
His Cys Ser Gin Lys Phe
60
Ser Ser Asn Thr Ala Tyr
145 JP 2004-071763
65 70 75 80
lie Gin Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ser Ser Gly Gly Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
10 115
<210> 149
<211> 112
15 <212> PRT
<213> Mus musculus
<400> 149
Asp Me Val Met Thr Gin Ala Ala Pro Ser Val Ser Val Thr Pro Gly
20 1 5 10 15
Glu Ser Val Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser
20 25 30
25 Asn Gly Asn Me Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
30
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Me
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
35 85 90 95
146 JP 2004-071763
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu I le Lys
100 105 110
5 <210> 150
<211> 118
<212> PRT
<213> Mus musculus
10 <400> 150
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
15 10 15
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Ser
15 20 25 30
Trp Met Asn Trp Val Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
35 40 45
20 Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn Gly Lys Phe
50 55 60
Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
25
Met Glu Me Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
30 100 105 110
Leu Val Thr Val Ser Ala
115
35
<210> 151
147 JP 2004-071763
<211> 112
<212> PRT
<213> Mus musculus
5 <400> 151
Asp He Val Met Thr Gin Ala Ala Pro Ser Val Pro Val Thr Pro Gly
15 10 15
G I u Ser Val Ser I le Ser Cys Arg Ser Ser Lys Ser Leu Leu His Asn
10 20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
15 Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me
65 70 75 80
20
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
Me Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
25 100 105 110
<210> 152
<211> 118
30 <212> PRT
<213> Mus musculus
<400> 152
Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
35 1 5 10 15
148 JP 2004-071763
Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Ser
20 25 30
Trp Met Asn Trp Val Asn Gin Arg Pro Gly Lys Gly Leu Glu Trp Me
5 35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Asp Thr Me Tyr Asn Gly Asn Phe
50 55 60
10 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Me Ala Tyr
65 70 75 80
Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
15
Thr Ser Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
20
Leu Val Thr Val Ser Ala
115
<210> 153
<211> 112
25 <212> PRT
<213> Mus musculus
<400> 1 53
Asp Me Val Met Thr Gin Ala Ala Pro Ser Leu Pro Val Thr Pro Gly
30 1 5 10 15
Glu Ser Val Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
35 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser
35 40 45
149 JP 2004-071763
Pro Gin Leu Leu Me Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
5 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg I le
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
10
Leu Glu Tyr Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
100 105 110
15 <210> 154
<211> 423
<212> DNA
<213> Mus musculus
20 <220>
<221> CDS
<222> (1) . . (423)
<223>
25 <400> 154
atg gtt ctt gcc age tct acc acc age ate cac ace atg ctg etc ctg 48
Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
15 10 15
30 etc ctg atg ctg gcc cag ccg gcc atg gcg gaa gtg aag ctg gtg gag 96
Leu Leu Met Leu Ala Gin Pro Ala Met Ala Glu Val Lys Leu Val Glu
20 25 30
35
tct ggg gga ggc tta gtg aag cct gga ggg tec egg aaa etc tec tgt
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Arg Lys Leu Ser Cys
35 40 45
144
150 JP 2004-071763
gca gcc tct gga ttc act ttc agt age tat acc atg tct tgg gtt cgc 192
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser Trp Val Arg
50 55 60
cag act ccg gcg aag agg ctg gag tgg gtc gca acc att agt agt ggc 240
Gin Thr Pro Ala Lys Arg Leu Glu Trp Val Ala Thr Me Ser Ser Gly
65 70 75 80
10 agt agt acc ate tac tat gca gac aca gtg aag ggc cga ttc acc ate 288
Ser Ser Thr He Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Me
85 90 95
tec aga gac aat gcc aag aac acc ctg ttc ctg caa atg acc agt eta 336
15 Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu Gin Met Thr Ser Leu
100 105 110
agg tct gag gac aca gcc atg tat tac tgt gca agg aga tgg ttt ctt 384
Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Trp Phe Leu
20 115 120 125
gac tgc tgg ggc caa ggc acc act etc aca gtc tec teg
Asp Cys Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
130 135 140
423
25
<210> 155
<211> 141
<212> PRT
30 <213> Mus musculus
<400> 155
Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
1 5 10 15
35
Leu Leu Met Leu Ala Gin Pro Ala Met Ala Glu Val Lys Leu Val Glu
151 JP 2004-071763
20 25 30
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Arg Lys Leu Ser Cys
35 40 45
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser Trp Val Arg
50 55 60
Gin Thr Pro Ala Lys Arg Leu Glu Trp Val Ala Thr Me Ser Ser Gly
10 65 70 75 80
Ser Ser Thr Me Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Me
85 90 95
15 Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu Gin Met Thr Ser Leu
100 105 110
Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Arg Trp Phe Leu
115 120 125
20
Asp Cys Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
130 135 140
25 <210> 156
<211> 357
<212> DNA
<213> Mus musculus
30 <220>
<221> CDS
<222> (1) . . (357)
<223>
35 <400> 156
gat att gtg etc acc caa tct cca get tct ttg get gtg tct eta ggg 48
152 JP 2004-071763
Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
15 10 15
cag agt gtc acc ate tec tgc aga gec agt gaa agt gtt gaa tat tat
5 Gin Ser Val Thr Me Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
96
ggc act agt tta atg cag tgg tac caa cag aaa cca gga cag cca ccc
Gly Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro
10 35 40 45
144
aaa etc etc ate tat ggt gca tec aac gta gaa tct ggg gtc cct gec
Lys Leu Leu lie Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
15
agg ttt agt ggc agt ggg tct ggg aca gac ttc age etc aac ate cat
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Me His
65 70 75 80
192
240
20 cct gtg gag gag gat gat att gca atg tat ttc tgt cag caa agt agg
Pro Val Glu Glu Asp Asp Me Ala Met Tyr Phe Cys Gin Gin Ser Arg
85 90 95
288
aag gtt ccg tgg acg ttc ggt gga ggc acc aag ctg gaa ata aag gac
25 Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Me Lys Asp
100 105 110
336
tac aag gat gac gac gat aag
Tyr Lys Asp Asp Asp Asp Lys
30 115
357
<210> 157
<211> 119
35 <212> PRT
<213> Mus musculus
153 JP 2004-071763
<400> 1 57
Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gin Ser Val Thr Me Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr
20 25 30
Gly Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro
10 35 40 45
Lys Leu Leu lie Tyr Gly Ala Ser Asn Val Glu Ser Gly Val Pro Ala
50 55 60
15 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Me His
65 70 75 80
Pro Val Glu Glu Asp Asp Me Ala Met Tyr Phe Cys Gin Gin Ser Arg
85 90 95
20
Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu I le Lys Asp
100 105 110
Tyr Lys Asp Asp Asp Asp Lys
25 115
<210> 158
<211> 432
30 <212> DNA
<213> Mus musculus
<220>
<221> CDS
35 <222> (1). . (432)
<223>
154
<400> 158
atg gtt ctt gcc age tct acc acc age ate cac ace atg ctg etc ctg
Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
15 10 15
etc ctg atg ctg gcc cag ccg gcc atg gcg cag gtt cag etc cag caa
Leu Leu Met Leu Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin
20 25 30
tct gga cct gag ctg gtg aag cct ggg gcc tea gtg aag att tec tgc
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Me Ser Cys
35 40 45
aag get tct ggc tat gca ttc agt age tec tgg atg aac tgg atg aag
Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser Trp Met Asn Trp Met Lys
50 55 60
cag agg cct gga aag ggt ctt gag tgg att ggg egg att tat cct gga
Gin Arg Pro Gly Lys Gly Leu Glu Trp Me Gly Arg Me Tyr Pro Gly
65 70 75 80
gat gga gat act aac tac aat ggg aag ttc aag ggc aag gcc aca ctg
Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
85 90 95
act gca gac aaa tec tec age aca gcc tac atg caa etc age age ctg
Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu
100 105 110
aca tct gag gac tct gcg gtc tac ttc tgt gca aga gcg agg aaa act
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Ala Arg Lys Thr
115 120 125
tec tgg ttt get tac tgg ggc caa ggg act ctg gtc act gtc tct gcg
Ser Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala
155 JP 2004-071763
130 135 140
<210> 159
5 <211> 144
<212> PRT
<213> Mus musculus
<400> 1 59
10 Met Val Leu Ala Ser Ser Thr Thr Ser Me His Thr Met Leu Leu Leu
15 10 15
Leu Leu Met Leu Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin
20 25 30
15
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Me Ser Cys
35 40 45
Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser Trp Met Asn Trp Met Lys
20 50 55 60
Gin Arg Pro Gly Lys Gly Leu Glu Trp Me Gly Arg lie Tyr Pro Gly
65 70 75 80
25 Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
85 90 95
Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu
100 105 110
30
Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Ala Arg Lys Thr
115 120 125
Ser Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala
35 130 135 140
156 JP 2004-071763
<210> 160
<211> 345
<212> DNA
5 <213> Mus musculus
<220>
<221> CDS
<222> (1). . (345)
10 <223>
<400> 1 60
gac att gtg ttg aca cag tct caa aaa ttc atg tec aca tea gta gga 48
Asp Me Val Leu Thr Gin Ser Gin Lys Phe Met Ser Thr Ser Val Gly
15 1 5 10 15
20
gac agg gtc age ate age tgc aag gee agt cag aat gtg ggt aat att 96
Asp Arg Val Ser lie Ser Cys Lys Ala Ser Gin Asn Val Gly Asn lie
20 25 30
ata gee tgg tat caa cag aaa cca ggg caa tct cct aaa gca ctg att 144
He Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Ala Leu Me
35 40 45
25 tac ttg gca tec tac egg tac agt gga gtc cct gat cgc ttc aca ggc 192
Tyr Leu Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
agt gga tct ggg aca gat ttc act etc acc att agt aat gtg cag tct 240
30 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Me Ser Asn Val Gin Ser
65 70 75 80
gaa gac ttg gca gag tat ttc tgt cag caa tat age age tct ccg etc 288
Glu Asp Leu Ala Glu Tyr Phe Cys Gin Gin Tyr Ser Ser Ser Pro Leu
35 85 90 95
157 JP 2004-071763
acg ttc ggt get ggg acc aag ctg gaa ata aag gac tac aag gat gac
Thr Phe Gly Ala Gly Thr Lys Leu Glu Me Lys Asp Tyr Lys Asp Asp
100 105 110
336
gac gat aag
Asp Asp Lys
115
345
10 <210> 161
<211> 115
<212> PRT
<213> Mus musculus
15 <400> 161
Asp lie Val Leu Thr Gin Ser Gin Lys Phe Met Ser Thr Ser Val Gly
15 10 15
Asp Arg Val Ser Me Ser Cys Lys Ala Ser Gin Asn Val Gly Asn Me
20 20 25 30
Me Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Ala Leu Me
35 40 45
25 Tyr Leu Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Me Ser Asn Val Gin Ser
65 70 75 80
30
Glu Asp Leu Ala Glu Tyr Phe Cys Gin Gin Tyr Ser Ser Ser Pro Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Me Lys Asp Tyr Lys Asp Asp
35 100 105 110
158 JP 2004-071763
Asp Asp Lys
115
5 <210> 162
<211> 116
<212> PRT
<213> Mus musculus
10 <400> 162
Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin
15 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Me Thr Ser Asp
15 20 25 30
Tyr Ala Trp Ser Trp Me Arg Gin Leu Pro Gly Asn Lys Leu Glu Trp
35 40 45
20 Met Gly Tyr Me Thr Tyr Ser Gly Tyr Ser Me Tyr Asn Pro Ser Leu
50 55 60
Lys Ser Arg Me Ser Me Ser Arg Asp Thr Ser Lys Asn Gin Leu Phe
65 70 75 80
25
Leu Gin Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Val Gly Gly Tyr Asp Asn Met Asp Tyr Trp Gly Gin Gly Thr Ser Val
30 100 105 110
Thr Val Ser Ser
115
35
<210> 163
159 JP 2004-071763
<211> 108
<212> PRT
<213> Mus musculus
5 <400> 1 63
Gin Me Val Leu Thr Gin Ser Pro Ala Me Met Ser Ala Ser Pro Gly
15 10 15
Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser
10 20 25 30
His Leu Tyr Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys Leu Trp
35 40 45
15 Me Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Me Ser Asn Met Glu
65 70 75 80
20
Thr Glu Asp Ala Ala Ser Tyr Phe Cys His Gin Trp Ser Ser Tyr Pro
85 90 95
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys
25 100 105
<210> 164
<211> 1924
30 <212> DNA
<213> Macaca fascicular is
<220>
<221> CDS
35 <222> (11).. (1918)
<223>
160
<400> 164
gaattccacc atg ccc tec tgg gec etc ttc atg gtc acc tec tgc etc
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu
1 5 10
etc ctg gee cct caa aac ctg gee caa gtc age age caa gat gtc tec
Leu Leu Ala Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser
15 20 25
ttg ctg gee teg gac tea gag ccc ctg aag tgt ttc tec cga aca ttt
Leu Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
30 35 40 45
gag gac etc act tgc ttc tgg gat gag gaa gag gca gca ccc agt ggg
Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly
50 55 60
aca tac cag ctg ctg tat gee tac ccg ggg gag aag ccc cgt gee tgc
Thr Tyr Gin Leu Leu Tyr Ala Tyr Pro Gly Glu Lys Pro Arg Ala Cys
65 70 75
ccc ctg agt tct cag age gtg ccc cgc ttt gga acc cga tac gtg tgc
Pro Leu Ser Ser Gin Ser Val Pro Arg Phe Gly Thr Arg Tyr Val Cys
80 85 90
cag ttt cca gee cag gaa gaa gtg cgt etc ttc tct ccg ctg cac etc
Gin Phe Pro Ala Gin Glu Glu Val Arg Leu Phe Ser Pro Leu His Leu
95 100 105
tgg gtg aag aat gtg ttc eta aac cag act cag att cag cga gtc etc
Trp Val Lys Asn Val Phe Leu Asn Gin Thr Gin Me Gin Arg Val Leu
110 115 120 125
ttt gtg gac agt gta ggc ctg ccg get ccc ccc agt ate ate aag gee
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Me Me Lys Ala
161
JP 2004-071763
130 135 140
atg ggt ggg age cag cca ggg gaa ctt cag ate age tgg gag gee cca 481
Met Gly Gly Ser Gin Pro Gly Glu Leu Gin Me Ser Trp Glu Ala Pro
5 145 150 155
get cca gaa ate agt gat ttc ctg agg tac gaa etc cgc tat ggc ccc 529
Ala Pro Glu Me Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro
160 165 170
10
aaa gat etc aag aac tec act ggt ccc acg gtc ata cag ttg ate gee 577
Lys Asp Leu Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu lie Ala
175 180 185
15 aca gaa acc tgc tgc cct get ctg cag agg cca cac tea gee tct get 625
Thr Glu Thr Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala
190 195 200 205
ctg gac cag tct cca tgt get cag ccc aca atg ccc tgg caa gat gga 673
20 Leu Asp Gin Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly
210 215 220
cca aag cag acc tec cca act aga gaa get tea get ctg aca gca gtg 721
Pro Lys Gin Thr Ser Pro Thr Arg Glu Ala Ser Ala Leu Thr Ala Val
25 225 230 235
ggt gga age tgc etc ate tea gga etc cag cct ggc aac tec tac tgg 769
Gly Gly Ser Cys Leu Me Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp
240 245 250
30
ctg cag ctg cgc age gaa cct gat ggg ate tec etc ggt ggc tec tgg 817
Leu Gin Leu Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp
255 260 265
35 gga tec tgg tec etc cct gtg act gtg gac ctg cct gga gat gca gtg 865
Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
162
JP 2004-071763
270 275 280 285
gca att gga ctg caa tgc ttt acc ttg gac ctg aag aat gtt acc tgt 913
Ala Me Gly Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys
5 290 295 300
caa tgg cag caa gag gac cat get agt tec caa ggt ttc ttc tac cac 961
Gin Trp Gin Gin Glu Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His
305 310 315
10
age agg gca egg tgc tgc ccc aga gac agg tac ccc ate tgg gag gac 1009
Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro I le Trp Glu Asp
320 325 330
15 tgt gaa gag gaa gag aaa aca aat cca gga tta cag acc cca cag ttc 1057
Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe
335 340 345
tct cgc tgc cac ttc aag tea cga aat gac age gtt att cac ate ctt 1105
20 Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser Val lie His Me Leu
350 355 360 365
gtg gag gtg acc aca gee ctg ggt get gtt cac agt tac ctg ggc tec 1153
Val Glu Val Thr Thr Ala Leu Gly Ala Val His Ser Tyr Leu Gly Ser
25 370 375 380
cct ttc tgg ate cac cag get gtg cgc etc ccc acc cca aac ttg cac 1201
Pro Phe Trp Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His
385 390 395
30
tgg agg gag ate tec age ggg cat ctg gaa ttg gag tgg cag cac cca 1249
Trp Arg Glu lie Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro
400 405 410
35 tea tec tgg gca gec caa gag acc tgc tat caa etc cga tac aca gga 1297
Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly
163
JP 2004-071763
415 420 425
gaa ggc cat cag gac tgg aag gtg ctg gag ccg cct etc ggg gec cga 1345
Glu Gly His Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg
5 430 435 440 445
gga ggg acc ctg gag ctg cgc ccg cga tct cgc tac cgt tta cag ctg 1393
Gly Gly Thr Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu
450 455 460
10
cgc gec agg etc aat ggc ccc acc tac caa ggt ccc tgg age teg tgg 1441
Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp
465 470 475
15 teg gac cca get agg gtg gag acc gee acc gag acc gee tgg att tec 1489
Ser Asp Pro Ala Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser
480 485 490
ttg gtg acc get ctg ctg eta gtg ctg ggc etc age gee gtc ctg ggc 1537
20 Leu Val Thr Ala Leu Leu Leu Val Leu Gly Leu Ser Ala Val Leu Gly
495 500 505
ctg ctg ctg ctg agg tgg cag ttt cct gca cac tac agg aga ctg agg 1585
Leu Leu Leu Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg
25 510 515 520 525
cat gee ctg tgg ccc tea ctt cca gat ctg cac cga gtc eta ggc cag 1633
His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin
530 535 540
30
tac ctt agg gac act gca gee ctg agt ccg ccc aag gee aca gtc tea 1681
Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser
545 550 555
35 gat acc tgt gaa gaa gtg gaa ccc age etc ctt gaa ate etc ccc aag 1729
Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Me Leu Pro Lys
164
JP 2004-071763
10
20
30
560 565 570
tec tea gag agg act cct ttg ccc ctg tgt tec tec cag tec cag atg 1777
Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ser Gin Met
5 575 580 585
gac tac cga aga ttg cag cct tct tgc ctg ggg ace atg ccc ctg tct 1825
Asp Tyr Arg Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser
590 595 600 605
gtg tgc cca ccc atg get gag tea ggg tec tgc tgt ace ace cac att 1873
Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me
610 615 620
15 gec aac cat tec tac eta cca eta age tat tgg cag cag cct tga 1918
Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
gtcgac
<210> 165
<211> 635
<212> PRT
25 <213> Macaca fascicular is
<400> 1 65
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
15 10 15
Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 25 30
1924
35
Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
35 40 45
165 JP 2004-071763
Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin
50 55 60
Leu Leu Tyr Ala Tyr Pro Gly Glu Lys Pro Arg Ala Cys Pro Leu Ser
5 65 70 75 80
Ser Gin Ser Val Pro Arg Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro
85 90 95
10 Ala Gin Glu Glu Val Arg Leu Phe Ser Pro Leu His Leu Trp Val Lys
100 105 110
Asn Val Phe Leu Asn Gin Thr Gin Me Gin Arg Val Leu Phe Val Asp
115 120 125
15
Ser Val Gly Leu Pro Ala Pro Pro Ser He lie Lys Ala Met Gly Gly
130 135 140
Ser Gin Pro Gly Glu Leu Gin He Ser Trp Glu Ala Pro Ala Pro Glu
20 145 150 155 160
e Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Lys Asp Leu
165 170 175
25 Lys Asn Ser Thr Gly Pro Thr Val I le Gin Leu I le Ala Thr Glu Thr
180 185 190
Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala Leu Asp Gin
1 95 200 205
30
Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly Pro Lys Gin
210 215 220
Thr Ser Pro Thr Arg Glu Ala Ser Ala Leu Thr Ala Val Gly Gly Ser
35 225 230 235 240
166 JP 2004-071763
Cys Leu lie Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu
245 250 255
Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp Gly Ser Trp
5 260 265 270
Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Me Gly
275 280 285
10 Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gin Trp Gin
290 295 300
Gin Glu Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala
305 310 315 320
15
Arg Cys Cys Pro Arg Asp Arg Tyr Pro Me Trp Glu Asp Cys Glu Glu
325 330 335
Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys
20 340 345 350
His Phe Lys Ser Arg Asn Asp Ser Val Me His Me Leu Val Glu Val
355 360 365
25 Thr Thr Ala Leu Gly Ala Val His Ser Tyr Leu Gly Ser Pro Phe Trp
370 375 380
Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu
385 390 395 400
30
lie Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp
405 410 415
Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His
35 420 425 430
167 JP 2004-071763
Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr
435 440 445
Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg
5 450 455 460
Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro
465 470 475 480
10 Ala Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser Leu Val Thr
485 490 495
Ala Leu Leu Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu
500 505 510
15
Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
515 520 525
Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg
20 530 535 540
Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys
545 550 555 560
25 Glu Glu Val Glu Pro Ser Leu Leu Glu lie Leu Pro Lys Ser Ser Glu
565 570 575
Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ser Gin Met Asp Tyr Arg
580 585 590
30
Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro
595 600 605
35
Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me Ala Asn His
610 615 620
168 JP 2004-071763
Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
5 <210> 166
<211> 24
<212> DNA
<213> Artificial
10 <220>
<223> an artificially synthesized sequence
<400> 1 66
caggggccag tggatagact gatg 24
15
<210> 167
<211> 23
<212> DNA
20 <213> Artificial
<220>
<223> an artificially synthesized sequence
25 <400> 167
gctcactgga tggtgggaag atg 23
<210> 168
30 <211> 30
<212> DNA
<213> Artificial
<220>
35 <223> an artificially synthesized primer sequence
169
JP 2004-071763
<400> 1 68
tagaattcca ccatggaatg gcctttgatc 30
5 <210> 169
<211> 56
<212> DNA
<213> Artificial
10 <220>
<223> an artificially synthesized primer sequence
<400> 1 69
agcctgagtc atcacaatat ccgatccgcc tccacctgca gagacagtga ccagag 56
15
<210> 170
<211> 56
<212> DNA
20 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
25 <400> 1 70
actctggtca ctgtctctgc aggtggaggc ggatcggata ttgtgatgac tcaggc 56
<210> 171
30 <211> 60
<212> DNA
<213> Artificial
<220>
35 <223> an artificially synthesized primer sequence
170 JP 2004-071763
25
<400> 171
attgcggccg cttatcactt atcgtcgtca tccttgtagt cttttatttc cagcttggtc 60
5 <210> 172
<211> 8
<212> PRT
<213> Artificial
10 <220>
<223> an artificially synthesized FLAG tag sequence
<400> 172
Asp Tyr Lys Asp Asp Asp Asp Lys
15 1 5
<210> 173
<211> 85
20 <212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 173
tagaattcca ccatggaatg gcctttgatc tttctcttcc tcctgtcagg aactgcaggt 60
gtccactccc aggttcagct gcagc 85
30
<210> 174
<211> 82
<212> DNA
35 <213> Artificial
171 JP 2004-071763
<220>
<223> an artificially synthesized primer sequence
<400> 1 74
5 tggtcactgt ctctgcaggt ggtggtggtt cgggtggtgg tggttcgggt ggtggcggat 60
cggatattgt gatgactcag gc 82
10 <210> 175
<211> 82
<212> DNA
<213> Artificial
15 <220>
<223> an artificially synthesized primer sequence
<400> 1 75
tgagtcatca caatatccga tccgccacca cccgaaccac caccacccga accaccacca 60
20
cctgcagaga cagtgaccag ag 82
<210> 176
25 <211> 25
<212> DNA
<213> Artificial
<220>
30 <223> an artificially synthesized primer sequence
<400> 1 76
caggttcagc tgcagcagtc tggac 25
35
<210> 177
172 JP 2004-071763
<211> 81
<212> DNA
<213> Artificial
5 <220>
<223> an artificially synthesized primer sequence
<400> 177
gctgcagctg aacctgcgat ccaccgcctc ccgaaccacc accacccgat ccaccacctc 60
10
cttttatttc cagcttggtc c 81
<210> 178
15 <211> 38
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 178
gcccagccgg ccatggcgga kgtrmagctt caggagtc 38
25
<210> 179
<211> 38
<212> DNA
<213> Artificial
30
<220>
<223> an artificially synthesized primer sequence
<400> 179
35 gcccagccgg ccatggcgga ggtbcagctb cagcagtc 38
173 JP 2004-071763
<210> 180
<211> 38
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 180
gcccagccgg ccatggcgca ggtgcagctg aagsastc 38
<210> 181
15 <211> 38
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 181
gcccagccgg ccatggcgga ggtccarctg caacartc 38
25
30
<210> 182
<211> 38
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
35
<400> 1 82
gcccagccgg ccatggcgca ggtycagctb cagcartc
38
174 JP 2004-071763
<210> 183
<211> 38
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 183
gcccagccgg ccatggcgca ggtycarctg cagcagtc 38
<210> 184
15 <211> 38
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 1 84
gcccagccgg ccatggcgca ggtccacgtg aagcagtc 38
25
30
<210> 185
<211> 38
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
35
<400> 1 85
gcccagccgg ccatggcgga ggtgaasstg gtggaatc
38
175 JP 2004-071763
<210> 186
<211> 38
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 186
gcccagccgg ccatggcgga vgtgawgytg gtggagtc 38
<210> 187
15 <211> 38
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 187
gcccagccgg ccatggcgga ggtgcagskg gtggagtc 38
<210> 188
<211> 38
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 1 88
35 gcccagccgg ccatggcgga kgtgcamctg gtggagtc 38
176
JP 2004-071763
<210> 189
<211> 38
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 189
gcccagccgg ccatggcgga ggtgaagctg atggartc 38
<210> 190
15 <211> 38
<212> DNA
<213> Artificial
<220>
20 <223> an artificial ly synthesized primer sequence
<400> 190
gcccagccgg ccatggcgga ggtgcarctt gttgagtc 38
25
30
<210> 191
<211> 38
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 191
35 gcccagccgg ccatggcgga rgtraagctt ctcgagtc 38
177
JP 2004-071763
<210> 192
<211> 38
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 192
gcccagccgg ccatggcgga agtgaarstt gaggagtc 38
<210> 193
15 <211> 40
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 1 93
gcccagccgg ccatggcgca ggttactctr aaagwgtstg 40
25
30
<210> 194
<211> 38
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
35
<400> 1 94
gcccagccgg ccatggcgca ggtccaactv cagcarcc
38
178 JP 2004-071763
<210> 195
<211> 38
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 195
gcccagccgg ccatggcgga tgtgaacttg gaagtgtc 38
<210> 196
15 <211> 38
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 196
gcccagccgg ccatggcgga ggtgaaggtc atcgagtc 38
25
<210> 197
<211> 36
<212> DNA
<213> Artificial
30
<220>
<223> an artificially synthesized primer sequence
<400> 1 97
35 ggagccgccg ccgcccgagg aaacggtgac cgtggt 36
179 JP 2004-071763
<210> 198
<211> 36
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 198
ggagccgccg ccgcccgagg agactgtgag agtggt 36
<210> 199
15 <211> 36
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 199
ggagccgccg ccgcccgcag agacagtgac cagagt 36
25
<210> 200
<211> 36
<212> DNA
<213> Artificial
30
<220>
<223> an artificially synthesized primer sequence
<400> 200
35 ggagccgccg ccgcccgagg agacggtgac tgaggt 36
180 JP 2004-071763
<210> 201
<211> 35
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 201
ggcggcggcg gctccgayat ccagctgact cagcc 35
<210> 202
15 <211> 35
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 202
ggcggcggcg gctccgayat tgttctcwcc cagtc 35
25
<210> 203
<211> 35
<212> DNA
<213> Artificial
30
<220>
<223> an artificially synthesized primer sequence
<400> 203
35 ggcggcggcg gctccgayat tgtgmtmact cagtc 35
181 JP 2004-071763
<210> 204
<211> 35
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 204
ggcggcggcg gctccgayat tgtgytraca cagtc 35
<210> 205
15 <211> 35
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 205
ggcggcggcg gctccgayat tgtratgacm cagtc 35
25
<210> 206
<211> 35
<212> DNA
<213> Artificial
30
<220>
<223> an artificially synthesized primer sequence
<400> 206
35 ggcggcggcg gctccgayat tmagatramc cagtc 35
182 JP 2004-071763
<210> 207
<211> 35
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 207
ggcggcggcg gctccgayat tcagatgayd cagtc 35
<210> 208
15 <211> 35
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 208
ggcggcggcg gctccgayat ycagatgaca cagac 35
<210> 209
<211> 35
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
35
<400> 209
ggcggcggcg gctccgayat tgttctcawc cagtc
35
f
183 JP 2004-071763
<210> 210
<211> 35
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 210
ggcggcggcg gctccgayat tgwgctsacc caatc 35
<210> 211
15 <211> 35
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 211
ggcggcggcg gctccgayat tstratgacc carte 35
25
30
<210> 212
<211> 35
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
35
<400> 212
ggcggcggcg gctccgayrt tktgatgacc carac
35
184 JP 2004-071763
<210> 213
<211> 35
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 213
ggcggcggcg gctccgayat tgtgatgacb cagkc 35
<210> 214
15 <211> 35
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 214
ggcggcggcg gctccgayat tgtgataacy cagga 35
<210> 215
<211> 35
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 215
35 ggcggcggcg gctccgayat tgtgatgacc cagwt 35
185 JP 2004-071763
<210> 216
<211> 35
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 216
ggcggcggcg gctccgayat tgtgatgaca caacc 35
<210> 217
15 <211> 35
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 217
ggcggcggcg gctccgayat tttgctgact cagtc 35
25
30
<210> 218
<211> 38
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
35
<400> 218
ggcggcggcg gctccgatgc tgttgtgact caggaatc
38
186
JP 2004-071763
<210> 219
<211> 36
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 219
ggaattcggc ccccgaggcc ttgatttcca gcttgg 36
<210> 220
15 <211> 36
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 220
ggaattcggc ccccgaggcc tttatttcca gcttgg 36
<210> 221
<211> 36
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized primer sequence
<400> 221
35 ggaattcggc ccccgaggcc tttatttcca actttg 36
187 JP 2004-071763
<210> 222
<211> 36
<212> DNA
5 <213> Artificial
<220>
<223> an artificially synthesized primer sequence
10 <400> 222
ggaattcggc ccccgaggcc ttcagctcca gcttgg 36
<210> 223
15 <211> 39
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized primer sequence
<400> 223
ggaattcggc ccccgaggcc cctaggacag tcagtttgg 39
25
<210> 224
<211> 27
<212> DNA
<213> Artificial
30
<220>
<223> an artificially synthesized sequence
<400> 224
35 ttactcgcgg cccagccggc catggcg 27
188
JP 2004-071763
<210> 225
<211> 17
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized sequence
10 <400> 225
ggaattcggc ccccgag 17
<210> 226
15 <211> 20
<212> DNA
<213> Artificial
<220>
20 <223> an artificially synthesized sequence
<400> 226
tcacttacag gctctctact 20
25
30
<210> 227
<211> 20
<212> DNA
<213> Artificial
<220>
<223> an artificially synthesized sequence
<400> 227
35 caggtggggt ctttcattcc 20
189 JP 2004-071763
<210> 228
<211> 354
<212> DNA
5 <213> Homo sapiens
<400> 228
caggtgcagc tggtgcagtc tggacctgag gtgaagaagc ctggggcctc agtgaaggtc 60
10 tcctgcaagg cttctggata caccttcacc aactcctgga tgaactgggt gaggcagagg 120
cctggaaagg gtcttgagtg gatgggacgg atttatcctg gagatggaga aactatctac 180
aatgggaaat tcagggtcag agtcacgatt accgcggacg aatccacgag cacagcctac 240
15
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggctat 300
gatgattact cgtttgctta ctggggccag ggaaccacgg tcaccgtctc ttca 354
20
<210> 229
<211> 118
<212> PRT
<213> Homo sapiens
25
<400> 229
Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
15 10 15
30 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser
20 25 30
Trp Met Asn Trp Val Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
35
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr I le
Tyr Asn Gly Lys Phe
190 JP 2004-071763
50 55 60
Arg Val Arg Val Thr He Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
5
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
10 100 105 110
Thr Val Thr Val Ser Ser
115
15
<210> 230
<211> 30
<212> PRT
<213> Homo sapiens
20
<400> 230
Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
1 5 10 15
25 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 231
30 <211> 5
<212> PRT
<213> Homo sapiens
<400> 231
35 Asn Ser Trp Met Asn
1 5
191 JP 2004-071763
<210> 232
<211> 14
5 <212> PRT
<213> Homo sapiens
<400> 232
Trp Val Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp Met Gly
10 1 5 10
<210> 233
<211> 17
15 <212> PRT
<213> Homo sapiens
<400> 233
Arg lie Tyr Pro Gly Asp Gly Glu Thr lie Tyr Asn Gly Lys Phe Arg
20 1 5 10 15
Val
25
<210> 234
<211> 32
<212> PRT
<213> Homo sapiens
30
<400> 234
Arg Val Thr lie Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu
15 10 15
35 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
192 JP 2004-071763
<210> 235
<211> 9
5 <212> PRT
<213> Homo sapiens
<400> 235
Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
10 1 5
<210> 236
<211> 11
15 <212> PRT
<213> Homo sapiens
<400> 236
Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser
20 1 5 10
<210> 237
<211> 336
25 <212> DNA
<213> Homo sapiens
<400> 237
gatattgtga tgactcagtc tgcactctcc ctgcccgtca cccctggaga gccggcctcc 60
30
atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 120
ttccagcaga agccagggca gtctccacag ctcctgatct atcggatgtc caaccttgcc 180
35 tcaggggtcc ctgacaggtt cagtggcagt ggatcaggca cagcttttac actgaaaatc 240
193 JP 2004-071763
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaacatat agaatatcct 300
tttacgttcg gccaagggac caaactggaa atcaaa 336
<210> 238
<211> 112
<212> PRT
<213> Homo sapiens
10
<400> 238
Asp He Val Met Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly
15 10 15
15 Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Gin Gin Lys Pro Gly Gin Ser
35 40 45
20
Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys I le
25 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
30 Me Glu Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu Me Lys
100 105 110
<210> 239
35 <211> 23
<212> PRT
194
JP 2004-071763
<213> Homo sapiens
<400> 239
Asp lie Val Met Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly
5 1 5 10 15
10
15
20
25
30
35
Glu Pro Ala Ser I le Ser Cys
20
<210> 240
<211> 16
<212> PRT
<213> Homo sapiens
<400> 240
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
<210> 241
<211> 15
<212> PRT
<213> Homo sapiens
<400> 241
Trp Phe Gin Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu Me Tyr
15 10 15
<210> 242
<211> 7
<212> PRT
<213> Homo sapiens
<400> 242
195 JP 2004-071763
Arg Met Ser Asn Leu Ala Ser
1 5
5 <210> 243
<211> 32
<212> PRT
<213> Homo sapiens
1 0 <400> 243
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr
15 10 15
Leu Lys Me Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
15 20 25 30
<210> 244
<211> 9
20 <212> PRT
<213> Homo sapiens
<400> 244
Met Gin His lie Glu Tyr Pro Phe Thr
25 1 5
<210> 245
<211> 10
30 <212> PRT
<213> Homo sapiens
<400> 245
Phe Gly Gin Gly Thr Lys Leu Glu Me Lys
35 1 5 10
r
196 JP 2004-071763
<210> 246
<211> 1924
<212> DNA
5 <213> Homo sapiens
<400> 246
gaattccacc atgccctcct gggccctctt
10 tcaaaacctg gcccaagtca gcagccaaga
cctgaagtgt ttctcccgaa catttgagga
agcgcccagt gggacatacc agctgctgta
15
ccccctgagt tcccagagca tgccccactt
ccaggaggaa gtgcgtctct tctttccgct
20 ccagactcgg actcagcgag tcctctttgt
tatcatcaag gccatgggtg ggagccagcc
agctccagaa atcagtgatt tcctgaggta
25
gaactccact ggtcccacgg tcatacagct
gcagagacct cactcagcct ctgctctgga
30 ctggcaagat ggaccaaagc agacctcccc
gggtggaagc tgcctcatct caggactcca
cagcgaacct gatgggatct ccctcggtgg
35
tgtggacctg cctggagatg cagtggcact
catggtcacc tcctgcctcc tcctggcccc 60
tgtctccttg ctggcatcag actcagagcc 120
cctcacttgc ttctgggatg aggaagaggc 180
tgcctacccg cgggagaagc cccgtgcttg 240
tggaacccga tacgtgtgcc agtttccaga 300
gcacctctgg gtgaagaatg tgttcctaaa 360
ggacagtgta ggcctgccgg ctccccccag 420
aggggaactt cagatcagct gggaggagcc 480
cgaactccgc tatggcccca gagatcccaa 540
gattgccaca gaaacctgct gccctgctct 600
ccagtctcca tgtgctcagc ccacaatgcc 660
aagtagagaa gcttcagctc tgacagcaga 720
gcctggcaac tcctactggc tgcagctgcg 780
ctcctgggga tcctggtccc tccctgtgac 840
tggactgcaa tgctttacct tggacctgaa 900
197
JP 2004-071763
gaatgttacc tgtcaatggc agcaacagga
cagcagggca cggtgctgcc ccagagacag
5
agagaaaaca aatccaggac tacagacccc
aaatgacagc attattcaca tccttgtgga
10 ctacctgggc tcccctttct ggatccacca
ctggagggag atctccagtg ggcatctgga
agcccaagag acctgttatc aactccgata
15
gctggagccg cctctcgggg cccgaggagg
ccgtttacag ctgcgcgcca ggctcaacgg
20 gtcggaccca actagggtgg agaccgccac
tctgcatcta gtgctgggcc tcagcgccgt
tcctgcacac tacaggagac tgaggcatgc
25
ggtcctaggc cagtacctta gggacactgc
agatacctgt gaagaagtgg aacccagcct
30 gactcctttg cccctgtgtt cctcccaggc
ttgcctgggg accatgcccc tgtctgtgtg
taccacccac attgccaacc attcctacct
35
cgac
ccatgctagc tcccaaggct tcttctacca 960
gtaccccatc tgggagaact gcgaagagga 1020
acagttctct cgctgccact tcaagtcacg 1080
ggtgaccaca gccccgggta ctgttcacag 1140
ggctgtgcgc ctccccaccc caaacttgca 1200
attggagtgg cagcacccat cgtcctgggc 1260
cacaggagaa ggccatcagg actggaaggt 1320
gaccctggag ctgcgcccgc gatctcgcta 1380
ccccacctac caaggtccct ggagctcgtg 1440
cgagaccgcc tggatctcct tggtgaccgc 1500
cctgggcctg ctgctgctga ggtggcagtt 1560
cctgtggccc tcacttccag acctgcaccg 1620
agccctgagc ccgcccaagg ccacagtctc 1680
ccttgaaatc ctccccaagt cctcagagag 1740
ccagatggac taccgaagat tgcagccttc 1800
cccacccatg gctgagtcag ggtcctgctg 1860
accactaagc tattggcagc agccttgagt 1920
1924
198 JP 2004-071763
<210> 247
<211> 1924
5 <212> DNA
<213> Homo sapiens
<220>
<221> CDS
10 <222> (11).. (1918)
<223>
<400> 247
gaattccacc atg ccc tec tgg gec etc ttc atg gtc acc tec tgc etc 49
15 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu
1 5 10
etc ctg gee cct caa aac ctg gee caa gtc age age caa gat gtc tec 97
Leu Leu Ala Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser
20 15 20 25
25
ttg ctg gca tea gac tea gag ccc ctg aag tgt ttc tec cga aca ttt 145
Leu Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
30 35 40 45
gag gac etc act tgc ttc tgg gat gag gaa gag gca gcg ccc agt ggg 193
Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly
50 55 60
30 aca tac cag ctg ctg tat gee tac ccg egg gag aag ccc cgt get tgc 241
Thr Tyr Gin Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys
65 70 75
35
ccc ctg agt tec cag age atg ccc cac ttt gga acc cga tac gtg tgc
Pro Leu Ser Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys
80 85 90
289
199
JP 2004-071763
cag ttt cca gac cag gag gaa gtg cct etc ttc ttt ccg ctg cac etc
Gin Phe Pro Asp Gin Glu Glu Val Pro Leu Phe Phe Pro Leu His Leu
95 100 105
337
tgg gtg aag aat gtg ttc eta aac cag act egg act cag cga gtc etc
Trp Val Lys Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu
110 115 120 125
385
10 ttt gtg gac agt gta ggc ctg ccg get ccc ccc agt ate ate aag gee
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Me Me Lys Ala
130 135 140
433
atg ggt ggg age cag cca ggg gaa ctt cag ate age tgg gag gag cca
15 Met Gly Gly Ser Gin. Pro Gly Glu Leu Gin Me Ser Trp Glu Glu Pro
145 150 155
481
get cca gaa ate agt gat ttc ctg agg tac gaa etc cgc tat ggc ccc
Ala Pro Glu Me Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro
20 160 165 170
529
25
aga gat ccc aag aac tec act ggt ccc acg gtc ata cag ctg att gee
Arg Asp Pro Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu Me Ala
175 180 185
aca gaa ace tgc tgc cct get ctg cag aga cct cac tea gee tct get
Thr Glu Thr Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala
190 195 200 205
577
625
30 ctg gac cag tct cca tgt get cag ccc aca atg ccc tgg caa gat gga
Leu Asp Gin Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly
210 215 220
673
cca aag cag acc tec cca agt aga gaa get tea get ctg aca gca gag
35 Pro Lys Gin Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu
225 230 235
721
200 JP 2004-071763
ggt gga age tgc etc ate tea gga etc cag cct ggc aac tec tac tgg 769
Gly Gly Ser Cys Leu lie Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp
240 245 250
5
ctg cag ctg cgc age gaa cct gat ggg ate tec etc ggt ggc tec tgg 817
Leu Gin Leu Arg Ser Glu Pro Asp Gly lie Ser Leu Gly Gly Ser Trp
255 260 265
10 gga tec tgg tec etc cct gtg act gtg gac ctg cct gga gat gca gtg 865
Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
270 275 280 285
gca ctt gga ctg caa tgc ttt ace ttg gac ctg aag aat gtt ace tgt 913
15 Ala Leu Gly Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys
290 295 300
caa tgg cag caa cag gac cat get age tec caa ggc ttc ttc tac cac 961
Gin Trp Gin Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His
20 305 310 315
age agg gca egg tgc tgc ccc aga gac agg tac ccc ate tgg gag aac 1009
Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Me Trp Glu Asn
320 325 330
25
tgc gaa gag gaa gag aaa aca aat cca gga eta cag ace cca cag ttc 1057
Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe
335 340 345
30 tct cgc tgc cac ttc aag tea cga aat gac age att att cac ate ctt 1105
Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser Me Me His Me Leu
350 355 360 365
35
gtg gag gtg ace aca gee ccg ggt act gtt cac age tac ctg ggc tec
Val Glu Val Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser
370 375 380
1153
201 JP 2004-071763
cct ttc tgg ate cac cag get gtg cgc etc ccc ace cca aac ttg cac
Pro Phe Trp Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His
385 390 395
1201
tgg agg gag ate tec agt ggg cat ctg gaa ttg gag tgg cag cac cca
Trp Arg Glu Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro
400 405 410
1249
10 teg tec tgg gca gee caa gag ace tgt tat caa etc cga tac aca gga
Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly
415 420 425
1297
gaa ggc cat cag gac tgg aag gtg ctg gag ccg cct etc ggg gee cga
15 Glu Gly His Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg
430 435 440 445
1345
gga ggg ace ctg gag ctg cgc ccg cga tct cgc tac cgt tta cag ctg
Gly Gly Thr Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu
20 450 455 460
1393
25
cgc gee agg etc aac ggc ccc ace tac caa ggt ccc tgg age teg tgg
Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp
465 470 475
teg gac cca act agg gtg gag ace gee ace gag acc gec tgg ate tec
Ser Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser
480 485 490
1441
1489
30 ttg gtg acc get ctg cat eta gtg ctg ggc etc age gee gtc ctg ggc
Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly
495 500 505
1537
ctg ctg ctg ctg agg tgg cag ttt cct gca cac tac agg aga ctg agg
35 Leu Leu Leu Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg
510 515 520 525
1585
202 JP 2004-071763
25
cat gcc ctg tgg ccc tea ctt cca gac ctg cac egg gtc eta ggc cag 1633
His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin
530 535 540
tac ctt agg gac act gca gcc ctg age ccg ccc aag gcc aca gtc tea 1681
Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser
545 550 555
10 gat acc tgt gaa gaa gtg gaa ccc age etc ctt gaa ate etc ccc aag 1729
Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Me Leu Pro Lys
560 565 570
tec tea gag agg act cct ttg ccc ctg tgt tec tec cag gcc cag atg 1777
15 Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met
575 580 585
gac tac cga aga ttg cag cct tct tgc ctg ggg acc atg ccc ctg tct 1825
Asp Tyr Arg Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser
20 590 595 600 605
gtg tgc cca ccc atg get gag tea ggg tec tgc tgt acc acc cac att 1873
Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me
610 615 620
gcc aac cat tec tac eta cca eta age tat tgg cag cag cct tga 1918
Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
30 gtcgac
1924
<210> 248
<211> 635
35 <212> PRT
<213> Homo sapiens
203 JP 2004-071763
<400> 248
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
15 10 15
Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 25 30
Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
10 35 40 45
Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin
50 55 60
15 Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys Pro Leu Ser
65 70 75 80
Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro
85 90 95
20
Asp Gin Glu Glu Val Pro Leu Phe Phe Pro Leu His Leu Trp Val Lys
100 105 110
Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu Phe Val Asp
25 115 120 125
Ser Val Gly Leu Pro Ala Pro Pro Ser He Me Lys Ala Met Gly Gly
130 135 140
30 Ser Gin Pro Gly Glu Leu Gin Me Ser Trp Glu Glu Pro Ala Pro Glu
145 150 155 160
e Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro
165 170 175
35
Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu Me Ala Thr Glu Thr
204 JP 2004-071763
180 185 190
Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala Leu Asp Gin
1 95 200 205
Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly Pro Lys Gin
210 215 220
Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser
10 225 230 235 240
Cys Leu Me Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu
245 250 255
15 Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp Gly Ser Trp
260 265 270
Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Leu Gly
275 280 285
20
Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gin Trp Gin
290 295 300
Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala
25 305 310 315 320
Arg Cys Cys Pro Arg Asp Arg Tyr Pro Me Trp Glu Asn Cys Glu Glu
325 330 335
30 Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys
340 345 350
His Phe Lys Ser Arg Asn Asp Ser Me Me His Me Leu Val Glu Val
355 360 365
35
Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser Pro Phe Trp
205 JP 2004-071763
370
375
380
I le His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu
385 390 395 400
He Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp
405 410 415
Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His
10 420 425 430
Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr
435 440 445
15 Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg
450 455 460
20
Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro
465 470 475 480
Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser Leu Val Thr
485 490 495
Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu
25 500 505 510
Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
515 520 525
30 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg
530 535 540
Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys
545 550 555 560
35
Glu Glu Val Glu Pro Ser Leu Leu Glu Me Leu Pro Lys Ser Ser Glu
15
20
25
30
206 JP 2004-071763
565 570 575
Arg Thr Pro Leu Pro Leu Cys Ser Ser 61 n Ala Gin Met Asp Tyr Arg
580 585 590
Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro
595 600 605
Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me Ala Asn His
10 610 615 620
Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
<210> 249
<211> 1924
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (11).. (1918)
<223>
<400> 249
gaattccacc atg ccc tec tgg gec etc ttc atg gtc acc tec tgc etc 49
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu
1 5 10
etc ctg gee cct caa aac ctg gec caa gtc age age caa gat gtc tec 97
Leu Leu Ala Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser
15 20 25
35 ttg ctg gca tea gac tea gag ccc ctg aag tgt ttc tec cga aca ttt 145
Leu Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
r
207 JP 2004-071763
10
30
30 35 40 45
gag gac etc act tgc ttc tgg gat gag gaa gag gca gcg ccc agt ggg 193
Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly
5 50 55 60
aca tac cag ctg ctg tat gec tac ccg egg gag aag ccc cgt get tgc 241
Thr Tyr Gin Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys
65 70 75
ccc ctg agt tec cag age atg ccc cac ttt gga ace cga tac gtg tgc 289
Pro Leu Ser Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys
80 85 90
15 cag ttt cca gac cag gag gaa gtg cgt etc ttc ttt ccg ctg cac etc 337
Gin Phe Pro Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu
95 100 105
tgg gtg aag aat gtg ttc eta aac cag act egg act cag cga gtc etc 385
20 Trp Val Lys Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu
110 115 120 125
ttt gtg gac agt gta ggc ctg ccg get ccc ccc agt ate ate aag gee 433
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Me Me Lys Ala
25 130 135 140
atg ggt ggg age cag cca ggg gaa ctt cag ate age tgg gag gag cca 481
Met Gly Gly Ser Gin Pro Gly Glu Leu Gin Me Ser Trp Glu Glu Pro
145 150 155
get cca gaa ate agt gat ttc ctg agg tac gaa etc cgc tat ggc ccc 529
Ala Pro Glu Me Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro
160 165 170
35 aga gat ccc aag aac tec act ggt ccc acg gtc ata cag ctg att gee 577
Arg Asp Pro Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu Me Ala
208
JP 2004-071763
175
180
185
aca gaa acc tgc tgc cct get ctg cag aga cct cac tea gee tct get
Thr Glu Thr Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala
5 190 195 200 205
625
10
ctg gac cag tct cca tgt get cag ccc aca atg ccc tgg caa gat gga
Leu Asp Gin Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly
210 215 220
cca aag cag acc tec cca agt aga gaa get tea get ctg aca gca gag
Pro Lys Gin Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu
225 230 235
673
721
15 ggt gga age tgc etc ate tea gga etc cag cct ggc aac tec tac tgg
Gly Gly Ser Cys Leu Me Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp
240 245 250
769
ctg cag ctg tgc age gaa cct gat ggg ate tec etc ggt ggc tec tgg
20 Leu Gin Leu Cys Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp
255 260 265
817
gga tec tgg tec etc cct gtg act gtg gac ctg cct gga gat gca gtg
Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
25 270 275 280 285
865
30
gca ctt gga ctg caa tgc ttt acc ttg gac ctg aag aat gtt acc tgt
Ala Leu Gly Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys
290 295 300
caa tgg cag caa cag gac cat get age tec caa ggc ttc ttc tac cac
Gin Trp Gin Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His
305 310 315
913
961
35 age agg gca egg tgc tgc ccc aga gac agg tac ccc ate tgg gag aac
Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Me Trp Glu Asn
1009
209 JP 2004-071763
320 325 330
tgc gaa gag gaa gag aaa aca aat cca gga eta cag acc cca cag ttc 1057
Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe
5 335 340 345
tct cgc tgc cac ttc aag tea cga aat gac age att att cac ate ctt 1105
Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser Me Me His Me Leu
350 355 360 365
10
gtg gag gtg acc aca gee ccg ggt act gtt cac age tac ctg ggc tec 1153
Val Glu Val Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser
370 375 380
15 cct ttc tgg ate cac cag get gtg cgc etc ccc acc cca aac ttg cac 1201
Pro Phe Trp Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His
385 390 395
tgg agg gag ate tec agt ggg cat ctg gaa ttg gag tgg cag cac cca 1249
20 Trp Arg Glu Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro
400 405 410
teg tec tgg gca gee caa gag acc tgt tat caa etc cga tac aca gga 1297
Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly
25 415 420 425
gaa ggc cat cag gac tgg aag gtg ctg gag ccg cct etc ggg gee cga 1345
Glu Gly His Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg
430 435 440 445
30
gga ggg acc ctg gag ctg cgc ccg cga tct cgc tac cgt tta cag ctg 1393
Gly Gly Thr Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu
450 455 460
35 cgc gee agg etc aac ggc ccc acc tac caa ggt ccc tgg age teg tgg 1441
Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp
210 JP 2004-071763
465 470 475
teg gac cca act agg gtg gag acc gec acc gag acc gec tgg ate tec 1489
Ser Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser
5 480 485 490
ttg gtg acc get ctg cat eta gtg ctg ggc etc age gee gtc ctg ggc 1537
Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly
495 500 505
10
ctg ctg ctg ctg agg tgg cag ttt cct gca cac tac agg aga ctg agg 1585
Leu Leu Leu Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg
510 515 520 525
15 cat gee ctg tgg ccc tea ctt cca gac ctg cac egg gtc eta ggc cag 1633
His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin
530 535 540
tac ctt agg gac act gca gec ctg age ccg ccc aag gec aca gtc tea 1681
20 Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser
545 550 555
gat acc tgt gaa gaa gtg gaa ccc age etc ctt gaa ate etc ccc aag 1729
Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Me Leu Pro Lys
25 560 565 570
tec tea gag agg act cct ttg ccc ctg tgt tec tec cag gee cag atg 1777
Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met
575 580 585
30
gac tac cga aga ttg cag cct tct tgc ctg ggg acc atg ccc ctg tct 1825
Asp Tyr Arg Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser
590 595 600 605
35 gtg tgc cca ccc atg get gag tea ggg tec tgc tgt acc acc cac att 1873
Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me
211 JP 2004-071763
610 615 620
gcc aac cat tec tac eta cca eta age tat tgg cag cag cct tga 1918
Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
5 625 630 635
gtcgac
10 <210> 250
<211> 635
<212> PRT
<213> Homo sapiens
15 <400> 250
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
1 5 10 15
Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 20 25 30
Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
35 40 45
25 Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin
50 55 60
Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys Pro Leu Ser
65 70 75 80
30
Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro
85 90 95
1924
Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys
35 100 105 110
212 JP 2004-071763
Asn Val Phe
115
Ser Val Gly
5 130
Ser Gin Pro
145
10 lie Ser Asp
Lys Asn Ser
15
Cys Cys Pro
195
Ser Pro Cys
20 210
Thr Ser Pro
225
25 Cys Leu lie
Cys Ser Glu
30
Ser Leu Pro
275
Leu Gin Cys
35 290
Leu Asn Gin Thr
Leu Pro Ala Pro
135
Gly Glu Leu Gin
150
Phe Leu Arg Tyr
165
Thr Gly Pro Thr
180
Ala Leu Gin Arg
Ala Gin Pro Thr
215
Ser Arg Glu Ala
230
Ser Gly Leu Gin
245
Pro Asp Gly Me
260
Val Thr Val Asp
Phe Thr Leu Asp
295
Arg Thr Gin Arg Val
120
Pro Ser Me Me Lys
140
I le Ser Trp Glu Glu
155
Glu Leu Arg Tyr Gly
170
Va I 1 1 e G I n Leu 1 1 e
185
Pro His Ser Ala Ser
200
Met Pro Trp Gin Asp
220
Ser Ala Leu Thr Ala
235
Pro Gly Asn Ser Tyr
250
Ser Leu Gly Gly Ser
265
Leu Pro Gly Asp Ala
280
Leu Lys Asn Val Thr
300
Leu Phe Val Asp
125
Ala Met Gly Gly
Pro Ala Pro Glu
160
Pro Arg Asp Pro
175
Ala Thr Glu Thr
190
Ala Leu Asp Gin
205
Gly Pro Lys Gin
Glu Gly Gly Ser
240
Trp Leu Gin Leu
255
Trp Gly Ser Trp
270
Val Ala Leu Gly
285
Cys Gin Trp Gin
213 JP 2004-071763
Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala
305 310 315 320
Arg Cys Cys Pro Arg Asp Arg Tyr Pro lie Trp Glu Asn Cys Glu Glu
5 325 330 335
Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys
340 345 350
10 His Phe Lys Ser Arg Asn Asp Ser Me Me His Me Leu Val Glu Val
355 360 365
Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser Pro Phe Trp
370 375 380
15
Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu
385 390 395 400
Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp
20 405 410 415
Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His
420 425 430
25 Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr
435 440 445
Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg
450 455 460
30
Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro
465 470 475 480
Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser Leu Val Thr
35 485 490 495
214 JP 2004-071763
Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu
500 505 510
Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
5 515 520 525
Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg
530 535 540
10 Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys
545 550 555 560
Glu Glu Val Glu Pro Ser Leu Leu Glu Me Leu Pro Lys Ser Ser Glu
565 570 575
15
Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met Asp Tyr Arg
580 585 590
Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro
20 595 600 605
Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His Me Ala Asn His
610 615 620
25 Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
<210> 251
30 <211> 1924
<212> DNA
<213> Homo sapiens
<220>
35 <221>
<222>
CDS
(11).. (1918)
215 JP 2004-071763
15
<223>
<400> 251
gaattccacc atg ccc tec tgg gec etc ttc atg gtc ace tec tgc etc 49
5 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu
1 5 10
etc ctg gee cct caa aac ctg gee caa gtc age age caa gat gtc tec 97
Leu Leu Ala Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser
10 15 20 25
ttg ctg gca tea gac tea gag ccc ctg aag tgt ttc tec cga aca ttt 145
Leu Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
30 35 40 45
gag gac etc act tgc ttc tgg gat gag gaa gag gca gcg ccc agt ggg 193
Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly
50 55 60
20 aca tac cag ctg ctg tat gee tac ccg egg gag aag ccc cgt get tgc 241
Thr Tyr Gin Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys
65 70 75
ccc ctg agt tec cag age atg ccc cac ttt gga ace cga tac gtg tgc 289
25 Pro Leu Ser Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys
80 85 90
cag ttt cca gac cag gag gaa gtg cgt etc ttc ttt ccg ctg cac etc 337
Gin Phe Pro Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu
30 95 100 105
tgg gtg aag aat gtg ttc eta aac cag act egg act cag cga gtc etc 385
Trp Val Lys Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu
110 115 120 125
35
ttt gtg gac agt gta ggc ctg ccg get ccc ccc agt ate ate aag gee
433
216 JP 2004-071763
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser lie Me Lys Ala
130 135 140
atg ggt ggg age cag cca ggg gaa ctt cag ate age tgg gag gag cca 481
5 Met Gly Gly Ser Gin Pro Gly Glu Leu Gin Me Ser Trp Glu Glu Pro
145 150 155
get cca gaa ate agt gat ttc ctg agg tac gaa etc cgc tat ggc ccc 529
Ala Pro Glu I le Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro
10 160 165 170
aga gat ccc aag aac tec act ggt ccc acg gtc ata cag ctg att gec 577
Arg Asp Pro Lys Asn Ser Thr Gly Pro Thr Val Me Gin Leu Me Ala
175 180 185
15
aca gaa acc tgc tgc cct get ctg cag aga cct cac tea gee tct get 625
Thr Glu Thr Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala
190 195 200 205
20 ctg gac cag tct cca tgt get cag ccc aca atg ccc tgg caa gat gga 673
Leu Asp Gin Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly
210 215 220
cca aag cag acc tec cca agt aga gaa get tea get ctg aca gca gag 721
25 Pro Lys Gin Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu
225 230 235
ggt gga age tgc etc ate tea gga etc cag cct ggc aac tec tac tgg 769
Gly Gly Ser Cys Leu I le Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp
30 240 245 250
ctg cag ctg cgc age gaa cct gat ggg ate tec etc ggt ggc tec tgg 817
Leu Gin Leu Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp
255 260 265
35
gga tec tgg tec etc act gtg act gtg gac ctg cct gga gat gca gtg 865
217
Gly Ser Trp Ser Leu Thr Val Thr Val Asp Leu Pro Gly Asp Ala Val
270 275 280 285
gca ctt gga ctg caa tgc ttt acc ttg gac ctg aag aat gtt acc tgt
Ala Leu Gly Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys
290 295 300
caa tgg cag caa cag gac cat get age tec caa ggc ttc ttc tac cac
Gin Trp Gin Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His
305 310 315
age agg gca egg tgc tgc ccc aga gac agg tac ccc ate tgg gag aac
Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro I le Trp Glu Asn
320 325 330
tgc gaa gag gaa gag aaa aca aat cca gga eta cag acc cca cag ttc
Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe
335 340 345
tct cgc tgc cac ttc aag tea cga aat gac age att att cac ate ctt
Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser Me Me His Me Leu
350 355 360 365
gtg gag gtg acc aca gee ccg ggt act gtt cac age tac ctg ggc tec
Val Glu Val Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser
370 375 380
cct ttc tgg ate cac cag get gtg cgc etc ccc acc cca aac ttg cac
Pro Phe Trp Me His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His
385 390 395
tgg agg gag ate tec agt ggg cat ctg gaa ttg gag tgg cag cac cca
Trp Arg Glu Me Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro
400 405 410
teg tec tgg gca gee caa gag acc tgt tat caa etc cga tac aca gga
218
Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly
415 420 425
gaa ggc cat cag gac tgg aag gtg ctg gag ccg cct etc ggg gec cga
5 Glu Gly His Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg
430 435 440 445
gga ggg acc ctg gag ctg cgc ccg cga tct cgc tac cgt tta cag ctg
Gly Gly Thr Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu
10 450 455 460
cgc gec agg etc aac ggc ccc acc tac caa ggt ccc tgg age teg tgg
Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp
465 470 475
15
teg gac cca act agg gtg gag acc gec acc gag acc gee tgg ate tec
Ser Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Me Ser
480 485 490
20 ttg gtg acc get ctg cat eta gtg ctg ggc etc age gec gtc ctg ggc
Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly
495 500 505
ctg ctg ctg ctg agg tgg cag ttt cct gca cac tac agg aga ctg agg
25 Leu Leu Leu Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg
510 515 520 525
cat gec ctg tgg ccc tea ctt cca gac ctg cac egg gtc eta ggc cag
His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin
30 530 535 540
tac ctt agg gac act gca gec ctg age ccg ccc aag gec aca gtc tea
Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser
545 550 555
35
gat acc tgt gaa gaa gtg gaa ccc age etc ctt gaa ate etc ccc aag
JP 2004-071763
1345
1393
1441
1489
1537
1585
1633
1681
1729
219 JP 2004-071763
15
Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu lie Leu Pro Lys
560 565 570
tec tea gag agg act cct ttg ccc ctg tgt tec tec cag gee cag atg 1777
5 Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met
575 580 585
gac tac cga aga ttg cag cct tct tgc ctg ggg acc atg ccc ctg tct 1825
Asp Tyr Arg Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser
10 590 595 600 605
gtg tgc cca ccc atg get gag tea ggg tec tgc tgt acc acc cac att 1873
Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His lie
610 615 620
gee aac cat tec tac eta cca eta age tat tgg cag cag cct tga 1918
Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
20 gtcgac
1924
<210> 252
<211> 635
25 <212> PRT
<213> Homo sapiens
<400> 252
Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala
30 1 5 10 15
Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala
20 25 30
35 Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu
35 40 45
220 JP 2004-071763
Thr Cys Phe
50
5 Leu Leu Tyr
65
Ser Gin Ser
10
Asp Gin Glu
Asn Val Phe
15 115
Ser Val Gly
130
20 Ser Gin Pro
145
1 1 e Ser Asp
25
Lys Asn Ser
Cys Cys Pro
30 195
Ser Pro Cys
210
Trp Asp Glu Glu
55
Ala Tyr Pro Arg
70
Met Pro His Phe
85
Glu Val Arg Leu
100
Leu Asn Gin Thr
Leu Pro Ala Pro
135
Gly Glu Leu Gin
150
Phe Leu Arg Tyr
165
Thr Gly Pro Thr
180
Ala Leu Gin Arg
Ala Gin Pro Thr
215
Glu Ala Ala Pro Ser
60
Glu Lys Pro Arg Ala
75
Gly Thr Arg Tyr Val
90
Phe Phe Pro Leu His
105
Arg Thr Gin Arg Val
120
Pro Ser Me Me Lys
140
I le Ser Trp Glu Glu
155
Glu Leu Arg Tyr Gly
170
Val Me Gin Leu Me
185
Pro His Ser Ala Ser
200
Met Pro Trp Gin Asp
220
Gly Thr Tyr Gin
Cys Pro Leu Ser
80
Cys Gin Phe Pro
95
Leu Trp Val Lys
110
Leu Phe Val Asp
125
Ala Met Gly Gly
Pro Ala Pro Glu
160
Pro Arg Asp Pro
175
Ala Thr Glu Thr
190
Ala Leu Asp Gin
205
Gly Pro Lys Gin
35 Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser
225 230 235 240
221 JP 2004-071763
Cys Leu I le Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu
245 250 255
5 Arg Ser Glu Pro Asp Gly Me Ser Leu Gly Gly Ser Trp Gly Ser Trp
260 265 270
Ser Leu Thr Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Leu Gly
275 280 285
10
Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gin Trp Gin
290 295 300
Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala
15 305 310 315 320
Arg Cys Cys Pro Arg Asp Arg Tyr Pro I le Trp Glu Asn Cys Glu Glu
325 330 335
20 Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys
340 345 350
His Phe Lys Ser Arg Asn Asp Ser Me Me His Me Leu Val Glu Val
355 360 365
25
Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser Pro Phe Trp
370 375 380
e His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu
30 385 390 395 400
e Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp
405 410 415
35 Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His
420 425 430
222 JP 2004-071763
Gin Asp Trp
435
5 Leu Glu Leu
450
Leu Asn Gly
465
10
Thr Arg Val
Ala Leu His
15
Leu Arg Trp
515
20 Trp Pro Ser
530
Asp Thr Ala
545
25
Glu Glu Val
Arg Thr Pro
30
Arg Leu Gin
595
35 Pro Met Ala
610
Lys Val Leu Glu
Arg Pro Arg Ser
455
Pro Thr Tyr Gin
470
Glu Thr Ala Thr
485
Leu Val Leu Gly
500
Gin Phe Pro Ala
Leu Pro Asp Leu
535
Ala Leu Ser Pro
550
Glu Pro Ser Leu
565
Leu Pro Leu Cys
580
Pro Ser Cys Leu
Glu Ser Gly Ser
615
Pro Pro Leu Gly Ala
440
Arg Tyr Arg Leu Gin
460
Gly Pro Trp Ser Ser
475
Glu Thr Ala Trp I le
490
Leu Ser Ala Val Leu
505
His Tyr Arg Arg Leu
520
His Arg Val Leu Gly
540
Pro Lys Ala Thr Val
555
Leu Glu Me Leu Pro
570
Ser Ser Gin Ala Gin
585
Gly Thr Met Pro Leu
600
Cys Cys Thr Thr His
620
Arg Gly Gly Thr
445
Leu Arg Ala Arg
Trp Ser Asp Pro
480
Ser Leu Val Thr
495
Gly Leu Leu Leu
510
Arg His Ala Leu
525
Gin Tyr Leu Arg
Ser Asp Thr Cys
560
Lys Ser Ser Glu
575
Met Asp Tyr Arg
590
Ser Val Cys Pro
605
Me Ala Asn His
223
Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro
625 630 635
<210> 253
<211> 1572
<212> DNA
<213> Homo sapiens
<400> 253
atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag
gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc
tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gcagaggcct
ggaaagggtc ttgagtgggt tggacggatt tatcctggag atggagaaac tatctacaat
gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat
gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt
ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctgca
ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt
ctcctgcata gtaatggcaa cacttacttg tattggtacc tgcagaagcc agggcagtct
ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt
ggcagtggat caggcacagc ttttacactg aaaatcagca gagtggaggc tgaggatgtt
ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa
224 JP 2004-071763
ctggaaatca aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840
gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 900
5
tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gcagaggcct 960
ggaaagggtc ttgagtgggt tggacggatt tatcctggag atggagaaac tatctacaat 1020
10 gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 1080
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 1140
gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 1200
15
ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctgca 1260
ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 1320
20 ctcctgcata gtaatggcaa cacttacttg tattggtacc tgcagaagcc agggcagtct 1380
ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 1440
ggcagtggat caggcacagc ttttacactg aaaatcagca gagtggaggc tgaggatgtt 1500
25
ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 1560
ctggaaatca aa 1572
30
<210> 254
<211> 524
<212> PRT
<213> Homo sapiens
35
<400> 254
225 JP 2004-071763
Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly
15 10 15
Val Gin Ser Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys
5 20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
10 Thr Asn Ser Trp Met Asn Trp Val Arg Gin Arg Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Gly Arg He Tyr Pro Gly Asp Gly Glu Thr He Tyr Asn
65 70 75 80
15
Gly Lys Phe Arg Val Arg Val Thr lie Thr Ala Asp Glu Ser Thr Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
20 100 105 110
Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
25 Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Val Met Thr Gin Ser Ala
145 150 155 160
30
Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser lie Ser Cys Arg
165 170 175
Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp
35 180 185 190
Tyr Leu Gin
195
Ser Asn Leu
5 210
Gly Thr Ala
225
10 Gly Val Tyr
Gin Gly Thr
15
Gly Ser Gly
275
Glu Val Lys
20 290
Gly Tyr Thr
305
25 Gly Lys Gly
Thr I le Tyr
30
Glu Ser Thr
355
Lys Pro Gly Gin
Ala Ser Gly Val
215
Phe Thr Leu Lys
230
Tyr Cys Met Gin
245
Lys Leu Glu lie
260
Gly Gly Gly Ser
Lys Pro Gly Ala
295
Phe Thr Asn Ser
310
Leu Glu Trp Val
325
Asn Gly Lys Phe
340
Ser Thr Ala Tyr
226
Ser Pro Gin Leu Leu
200
Pro Asp Arg Phe Ser
220
I le Ser Arg Val Glu
235
His I le Glu Tyr Pro
250
Lys Gly Gly Gly Gly
265
Gin Val Gin Leu Val
280
Ser Val Lys Val Ser
300
Trp Met Asn Trp Val
315
Gly Arg I le Tyr Pro
330
Arg Val Arg Val Thr
345
Met Glu Leu Ser Ser
360
JP 2004-071763
Me Tyr Arg Met
205
Gly Ser Gly Ser
Ala Glu Asp Val
240
Phe Thr Phe Gly
255
Ser Gly Gly Gly
270
Gin Ser Gly Pro
285
Cys Lys Ala Ser
Arg Gin Arg Pro
320
Gly Asp Gly Glu
335
I le Thr Ala Asp
350
Leu Arg Ser Glu
365
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe
35 370 375 380
227
JP 2004-071763
Ala Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
385 390 395 400
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He Val Met
5 405 410 415
Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser
420 425 430
10 lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr
435 440 445
Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu
450 455 460
15
lie Tyr Arg Met Ser Ash Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
465 470 475 480
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys lie Ser Arg Val Glu
20 485 490 495
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His He Glu Tyr Pro
500 505 510
25 Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys
515 520
<210> 255
30 <211> 354
<212> DNA
<213> Homo sapiens
<400> 255
35 caggtgcagc tggtgcagtc tggacctgag gtgaagaagc ctggggcctc agtgaaggtc
60
228
JP 2004-071763
tcctgcaagg cttctggata caccttcacc aactcctgga tgaactgggt gaggcagagg 120
cctggaaagg gtcttgagtg ggttggacgg atttatcctg gagatggaga aactatctac 180
5 aatgggaaat tcagggtcag agtcacgatt accgcggacg aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggctat 300
gatgattact cgtttgctta ctggggccag ggaaccacgg tcaccgtctc ttca 354
10
<210> 256
<211> 118
<212> PRT
15 <213> Homo sapiens
<400> 256
Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
15 10 15
20
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser
20 25 30
Trp Met Asn Trp Val Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp Val
25 35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr He Tyr Asn Gly Lys Phe
50 55 60
30 Arg Val Arg Val Thr Me Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
35
Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
229 JP 2004-071763
20
25
30
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 257
<211> 336
<212> DNA
10 <213> Homo sapiens
<400> 257
gatattgtga tgactcagtc tgcactctcc ctgcccgtca cccctggaga gccggcctcc 60
15 atctcctgca ggtctagtaa gagtctcctg catagtaatg gcaacactta cttgtattgg 120
tacctgcaga agccagggca gtctccacag ctcctgatct atcggatgtc caaccttgcc 180
tcaggggtcc ctgacaggtt cagtggcagt ggatcaggca cagcttttac actgaaaatc 240
agcagagtgg aggctgagga tgttggggtt tattactgca tgcaacatat agaatatcct 300
tttacgttcg gccaagggac caaactggaa atcaaa 336
<210> 258
<211> 112
<212> PRT
<213> Homo sapiens
<400> 258
Asp lie Val Met Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly
15 10 15
35 Glu Pro Ala Ser Me Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30
230 JP 2004-071763
Asn Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Ser
35 40 45
5 Pro Gin Leu Leu lie Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Me
65 70 75 80
10
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His
85 90 95
lie Glu Tyr Pro Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys
15 100 105 110
<210> 259
<211> 1572
20 <212> DNA
<213> Homo sapiens
<400> 259
atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag 60
25
gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120
tgcaaggctt ctggatacac cttcaccaac tcctggatga actggatcag gcagaggcct 180
30 ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240
gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 300
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 360
35
gattactcgt ttgcttactg gggccaggga accctggtca ccgtctcttc aggtggtggt 420
231 JP 2004-071763
ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctgca 480
ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 540
5
ctcctgcata gtaatggcaa cacttacttg tattggtacc tgcagaagcc agggcagtct 600
ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 660
10 ggcagtggat caggcacagc ttttacactg aaaatcagca gagtggaggc tgaggatgtt 720
ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 780
ctggaaatca aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840
15
gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 900
tgcaaggctt ctggatacac cttcaccaac tcctggatga actggatcag gcagaggcct 960
20 ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 1020
gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 1080
gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 1140
25
gattactcgt ttgcttactg gggccaggga accctggtca ccgtctcttc aggtggtggt 1200
ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctgca 1260
30 ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 1320
ctcctgcata gtaatggcaa cacttacttg tattggtacc tgcagaagcc agggcagtct 1380
ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 1440
35
ggcagtggat caggcacagc ttttacactg aaaatcagca gagtggaggc tgaggatgtt 1500
232 JP 2004-071763
ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 1560
ctggaaatca aa 1572
5
<210> 260
<211> 524
<212> PRT
10 <213> Homo sapiens
<400> 260
Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly
15 10 15
15
Val Gin Ser Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys
20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
20 35 40 45
Thr Asn Ser Trp Met Asn Trp lie Arg Gin Arg Pro Gly Lys Gly Leu
50 55 60
25 Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr lie Tyr Asn
65 70 75 80
Gly Lys Phe Arg Val Arg Val Thr Me Thr Ala Asp Glu Ser Thr Ser
85 90 95
30
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
35 115 120 125
233 JP 2004-071763
Gin Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Asp Me Val Met Thr Gin Ser Ala
5 145 150 155 160
Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Me Ser Cys Arg
165 170 175
10 Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp
180 185 190
Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu Me Tyr Arg Met
195 200 205
15
Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
210 215 220
Gly Thr Ala Phe Thr Leu Lys lie Ser Arg Val Glu Ala Glu Asp Val
20 225 230 235 240
Gly Val Tyr Tyr Cys Met Gin His Me Glu Tyr Pro Phe Thr Phe Gly
245 250 255
25 Gin Gly Thr Lys Leu Glu Me Lys Gly Gly Gly Gly Ser Gly Gly Gly
260 265 270
Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Val Gin Ser Gly Pro
275 280 285
30
Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser
290 295 300
Gly Tyr Thr Phe Thr Asn Ser Trp Met Asn Trp Me Arg Gin Arg Pro
35 305 310 315 320
234 JP 2004-071763
Gly Lys Gly Leu Glu Trp lie Gly Arg He Tyr Pro Gly Asp Gly Glu
325 330 335
Thr He Tyr Asn Gly Lys Phe Arg Val Arg Val Thr He Thr Ala Asp
5 340 345 350
Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
355 360 365
10 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe
370 375 380
Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
385 390 395 400
15
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He Val Met
405 410 415
Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser
20 420 425 430
lie Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr
435 440 445
25 Tyr Leu Tyr Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu
450 455 460
He Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
465 470 475 480
30
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys He Ser Arg Val Glu
485 490 495
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His lie Glu Tyr Pro
35 500 505 510
235 JP 2004-071763
20
35
Phe Thr Phe Gly Gin Gly Thr Lys Leu Glu He Lys
515 520
5 <210> 261
<211> 354
<212> DNA
<213> Homo sapiens
10 <400> 261
caggtgcagc tggtgcagtc tggacctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggata caccttcacc aactcctgga tgaactggat caggcagagg 120
15 cctggaaagg gtcttgagtg gattggacgg atttatcctg gagatggaga aactatctac 180
aatgggaaat tcagggtcag agtcacgatt accgcggacg aatccacgag cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagaggctat 300
gatgattact cgtttgctta ctggggccag ggaaccctgg tcaccgtctc ttca 354
<210> 262
25 <211> 118
<212> PRT
<213> Homo sapiens
<400> 262
30 Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
15 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser
20 25 30
Trp Met Asn Trp I le Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp I le
236 JP 2004-071763
35 40 45
Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr lie Tyr Asn Gly Lys Phe
50 55 60
5
Arg Val Arg Val Thr Me Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
10 85 90 95
Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gin Gly Thr
100 105 110
15 Leu Val Thr Val Ser Ser
115
<210> 263
20 <211> 1572
<212> DNA
<213> Mus musculus
<400> 263
25 atggaatggc ctttgatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactcccag 60
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 120
tgcaaggctt ctggctatgc attcactaac tcctggatga actgggtgaa gcagaggcct 180
30
ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240
gggaaattca gggtcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300
35 gatatcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aggctatgat 360
237 JP 2004-071763
gattactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc aggtggtggt 420
ggttcgggtg gtggtggttc gggtggtggc ggatcggata ttgtgatgac tcaggctgca 480
5 ccctctatac ctgtcactcc tggagagtca gtatccatct cctgtaggtc tagtaagagt 540
ctcctgcata gtaatggcaa cacttacttg tattggttcc tgcagaggcc aggccagtct 600
cctcaactcc tgatatatcg gatgtccaac cttgcctcag gagtcccaga taggttcagt 660
10
ggcagtgggt caggaactgc tttcacactg agaatcagta gagtggaggc tgaggatgtg 720
ggtgtttatt actgtatgca acatatagaa tatcctttta cgttcggatc ggggaccaag 780
15 ctggaaataa aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcctcagt gaagatttcc 900
tgcaaggctt ctggctatgc attcactaac tcctggatga actgggtgaa gcagaggcct 960
20
ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 1020
gggaaattca gggtcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 1080
25 gatatcagca gcctgacatc tgaggactct gcggtctact tctgtgcaag aggctatgat 1140
gattactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc aggtggtggt 1200
ggttcgggtg gtggtggttc gggtggtggc ggatcggata ttgtgatgac tcaggctgca 1260
30
ccctctatac ctgtcactcc tggagagtca gtatccatct cctgtaggtc tagtaagagt 1320
ctcctgcata gtaatggcaa cacttacttg tattggttcc tgcagaggcc aggccagtct 1380
35 cctcaactcc tgatatatcg gatgtccaac cttgcctcag gagtcccaga taggttcagt 1440
238 JP 2004-071763
ggcagtgggt caggaactgc tttcacactg agaatcagta gagtggaggc tgaggatgtg 1500
ggtgtttatt actgtatgca acatatagaa tatcctttta cgttcggatc ggggaccaag 1560
5 ctggaaataa aa 1572
<210> 264
<211> 524
10 <212> PRT
<213> Mus musculus
<400> 264
Met Glu Trp Pro Leu Me Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 1 5 10 15
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
20 Pro Gly Ala Ser Val Lys Me Ser Cys Lys Ala Ser Gly Tyr Ala Phe
35 40 45
Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro Gly Lys Gly Leu
50 55 60
25
Glu Trp Me Gly Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn
65 70 75 80
Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
30 85 90 95
Thr Ala Tyr Met Asp I le Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
35
Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly
115 120 125
239 JP 2004-071763
Gin Gly Thr
130
5 Gly Gly Ser
145
Pro Ser Me
10
Ser Ser Lys
Phe Leu Gin
15 195
Ser Asn Leu
210
20 Gly Thr Ala
225
Gly Val Tyr
25
Ser Gly Thr
Gly Ser Gly
30 275
Glu Leu Val
290
Leu Val Thr Val
135
Gly Gly Gly Gly
150
Pro Val Thr Pro
165
Ser Leu Leu His
180
Arg Pro Gly Gin
Ala Ser Gly Val
215
Phe Thr Leu Arg
230
Tyr Cys Met Gin
245
Lys Leu Glu Me
260
Gly Gly Gly Ser
Lys Pro Gly Ala
295
Ser Ala Gly Gly Gly
140
Ser Asp 1 1 e Va I Met
155
Gly Glu Ser Val Ser
170
Ser Asn Gly Asn Thr
185
Ser Pro Gin Leu Leu
200
Pro Asp Arg Phe Ser
220
Me Ser Arg Val Glu
235
His Me Glu Tyr Pro
250
Lys Gly Gly Gly Gly
265
Gin Val Gin Leu Gin
280
Ser Val Lys Me Ser
300
Gly Ser Gly Gly
Thr Gin Ala Ala
160
I le Ser Cys Arg
175
Tyr Leu Tyr Trp
190
I le Tyr Arg Met
205
Gly Ser Gly Ser
Ala Glu Asp Val
240
Phe Thr Phe Gly
255
Ser Gly Gly Gly
270
Gin Ser Gly Pro
285
Cys Lys Ala Ser
35 Gly Tyr Ala Phe Thr Asn Ser Trp Met Asn Trp Val Lys Gin Arg Pro
305 310 315 320
240 JP 2004-071763
Gly Lys Gly Leu Glu Trp Me Gly Arg lie Tyr Pro Gly Asp Gly Glu
325 330 335
5 Thr Me Tyr Asn Gly Lys Phe Arg Val Lys Ala Thr Leu Thr Ala Asp
340 345 350
Lys Ser Ser Ser Thr Ala Tyr Met Asp He Ser Ser Leu Thr Ser Glu
355 360 365
10
Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe
370 375 380
Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly
15 385 390 395 400
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Val Met
405 410 415
20 Thr Gin Ala Ala Pro Ser Me Pro Val Thr Pro Gly Glu Ser Val Ser
420 425 430
e Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr
435 440 445
25
Tyr Leu Tyr Trp Phe Leu Gin Arg Pro Gly Gin Ser Pro Gin Leu Leu
450 455 460
e Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser
30 465 470 475 480
Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Me Ser Arg Val Glu
485 490 495
35 Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin His Me Glu Tyr Pro
500 505 510
241 JP 2004-071763
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Me Lys
515 520
5
<210> 265
<211> 30
<212> PRT
<213> Homo sapiens
10
<400> 265
Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
15 10 15
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
<210> 266
20 <211> 5
<212> PRT
<213> Homo sapiens
<400> 266
25 Asn Ser Trp Met Asn
1 5
<210> 267
30 <211> 14
<212> PRT
<213> Homo sapiens
<400> 267
35 Trp Val Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp Val Gly
1 5 10
242 JP 2004-071763
<210> 268
<211> 17
5 <212> PRT
<213> Homo sapiens
<400> 268
Arg lie Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn Gly Lys Phe Arg
10 1 5 10 15
Val
15
<210> 269
<211> 32
<212> PRT
<213> Homo sapiens
20
<400> 269
Arg Val Thr He Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu
15 10 15
25 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 270
30 <211> 9
<212> PRT
<213> Homo sapiens
<400> 270
35 Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
243 JP 2004-071763
<210> 271
<211> 11
5 <212> PRT
<213> Homo sapiens
<400> 271
Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser
10 1 5 10
<210> 272
<211> 23
15 <212> PRT
<213> Homo sapiens
<400> 272
Asp He Val Met Thr Gin Ser Ala Leu Ser Leu Pro Val Thr Pro Gly
20 1 5 10 15
25
30
Glu Pro Ala Ser He Ser Cys
20
<210> 273
<211> 16
<212> PRT
<213> Homo sapiens
<400> 273
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr
15 10 15
35
<210> 274
244 JP 2004-071763
<211> 15
<212> PRT
<213> Homo sapiens
5 <400> 274
Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Gin Leu Leu lie Tyr
15 10 15
10 <210> 275
<211> 7
<212> PRT
<213> Homo sapiens
15 <400> 275
Arg Met Ser Asn Leu Ala Ser
1 5
20 <210> 276
<211> 32
<212> PRT
<213> Homo sapiens
25 <400> 276
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr
15 10 15
Leu Lys Me Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
30 20 25 30
<210> 277
<211> 9
35 <212> PRT
<213> Homo sapiens
245
JP 2004-071763
<400> 277
Met Gin His Me Glu Tyr Pro Phe Thr
1 5
<210> 278
<211> 10
<212> PRT
10 <213> Homo sapiens
<400> 278
Phe Gly Gin Gly Thr Lys Leu Glu lie Lys
1 5 10
<210> 279
<211> 30
<212> PRT
20 <213> Homo sapiens
<400> 279
Gin Val Gin Leu Val Gin Ser Gly Pro Glu Val Lys Lys Pro Gly Ala
15 10 15
25
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30
30 <210> 280
<211> 5
<212> PRT
<213> Homo sapiens
35 <400> 280
Asn Ser Trp Met Asn
246
JP 2004-071763
<210> 281
5 <211> 14
<212> PRT
<213> Homo sapiens
<400> 281
10 Trp Me Arg Gin Arg Pro Gly Lys Gly Leu Glu Trp lie Gly
1 5 10
<210> 282
15 <211> 17
<212> PRT
<213> Homo sapiens
<400> 282
20 Arg Me Tyr Pro Gly Asp Gly Glu Thr Me Tyr Asn Gly Lys Phe Arg
15 10 15
25
35
Val
<210> 283
<211> 32
<212> PRT
30 <213> Homo sapiens
<400> 283
Arg Val Thr Me Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu
15 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
247
JP 2004-071763
20 25 30
<210> 284
5 <211> 9
<212> PRT
<213> Homo sapiens
<400> 284
10 Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr
1 5
<210> 285
15 <211> 11
<212> PRT
<213> Homo sapiens
<400> 285
20 Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser
1 5 10
JP 2004-071763
[Document Name] Drawings
[Fig. 1]
dbVB22B
SIGNAL SEQUENCE L I N KE R (Gly 4 Ser)
*4 B
FLAG SEQUENCE
< 1- D
PvuO
i
T
Not I
PCR RECOMBINATION
scVB22B
VtM
VL
SIGNAL SEQUENCE
Pvu D
LINKER(Gly 4 Ser) 3
PCR AMPLIFICATION
FLAG SEQUENCE
F
Pvu n
VL
VB22B
SC(F\/)2 SIGNAL SEQUENCE LINKER(Gfy 4 Ser) 3
CLONING INTO
Pvull SITE
Pvu D
i
VH
LINKER (Gly 4 Ser) 3
LINKER(Gly 4 Sar) 3
VL
FLAG SEQUENCE
[Fig- 2]
CHO-HUMAN Mpl
■ • i t a i hi -i i i. i i ■ til i < | i
10" 10i iQ2
CHO-MQNKEY Mpl
■l ij • i a i 1 1 ill
i i i i 1 1 1 1
103
10°
10i
| I I I » 1 ll)|
10 2
103
FITC LOG
FITC LOG
249
JP 2004-071763
[Fig. 3]
1.6
0.000 0.001 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
[Fig.4]
1.8
0.000 0.001 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
JP 2004-071763
[Fig. 5]
to
uo
CO
ID
LU
O
<
CD
a:
o
CO
CQ
<
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.0001 0.001
0.01
0.1
1
10
100
CONCENTRATION (nM)
[Fig. 6]
CDRl
CDR2
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB4 5B
VB83
VBI15
VB14B
VB22B
VBI6
VBI57
VB43
VB51
VA7
VA130
VA2 59
VB17B
VB12B
VBI40
VB33
VB4 5B
VB83
VBI15
VBI4B
VB22B
VBI6
VB157
VB43
VB51
DIVMTQAAPSIPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVT PGESVS ISC
D I VMTQAAPS VPVT PGESVS I SC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVTPGESVSISC
D I VMTQAAPS VPVT PGE S VS ISC
D I VMTQAAPS I PVT PGE S VS ISC
DIVMTQAAPSVPVTPGESVSISC
D I VMTQAAPS VSVT PGESVS ISC
DIVMTQAAPSVPVT PGESVS ISC
D I VMTQAAPS LPVT PGESVS I SC
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNIYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNTYLY
RSSKSLLYSNGNIYLY
RSSKSLLHNNGNTYLY
RSSKSLLHSNGNTYLY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFMQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQS PQLLI Y
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
CDR3
GVPDRFSGSGSGTAFTLRI SRVEAEDVGIYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GAPDRFSGSGSGTAFTLRISRVETEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGAAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGAAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLTISSVEAEDVGVYYC
GVPDRFSGSGSGTAFTLKI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
GVPDRFSGSGSGTAFTLRI SRVEAEDVGVYYC
MQHLEYPFT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHVEYPYT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
FGTGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
251 JP 2004-071763
[Fig. 7]
VA7
VA130
VA259
VB17B
VB12B
VB140
VB33
VB4 5B
VB8B
VB115
VB14B
VB22B
VB16
VB157
VB4B
VB51
VA7
VA130
VA2 59
VB17B
VB12B
VB14 0
VB33
VB4 5B
VB8B
VB115
VB14B
VB22B
VB16
VB157
VB4B
VB51
D I VMTQAAP S I PVT PGE S VS I S C
DIVMTQAAPSVPVTPGESVSISC
DIVMTQAAPSVPVT PGESVS I SC
D IVMTQAAPSVPVT PGE S VS ISC
D I VMTQAAPSVPVT PGE S VS ISC
D IVMTQAAPSVPVT PGE S VS ISC
D IVMTQAAPSVPVT PGE S VS ISC
D IVMTQAAPSVPVT PGE S VS ISC
D IVMTQAAPSVPVT PGE SVS ISC
D IVMTQAAPSVPVT PGE SVS ISC
D IVMTQAAPSVPVT PGE SVS ISC
D I VMTQAAPS I PVT PGE SVS ISC
D IVMTQAAPSVPVT PGE SVS ISC
D IVMTQAAPSVSVT PGE SVS ISC
D IVMTQAAPSVPVT PGE SVS ISC
D I VMTQAAPS L PVT PGE SVS I S C
CDR1
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNIYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLHSNGNTYLY
RSSKSLLYSNGNTYLY
RSSKSLLYSNGNIYLY
RSSKSLLHNNGNTYLY
RSSKSLLHSNGNTYLY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFMQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
WFLQRPGQSPQLLIY
CDR3
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GAPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGAAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGAAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLTI
GVPDRFSGSGSGTAFTLKI
GVPDRFSGSGSGTAFTLRI
GVPDRFSGSGSGTAFTLRI
SRVEAEDVGIYYC
SRVEAEDVGVYYC
SRVETEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
S RVEAE D VG V Y YC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
SSVEAEDVGVYYC
S RVEAE D VG V Y YC
SRVEAEDVGVYYC
SRVEAEDVGVYYC
MQHLEYPFT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHLEYPYT
MQHVEYPYT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
MQHLEYPYT
MQHIEYPFT
MQHLEYPYT
FGTGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
FGSGTKLEIK
CDR2
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
RMSNLAS
252
JP 2004-071763
[Fig. 8]
CHO-HUMAN Mpl
CHO-MONKEY Mpl
.i i i i i i
FITC LOG
1 1 1 i i i i i i 1 1 1 i - i i i i 1 1 1 1 i i i i i 1 1 1 1
10° 10 1 10 2 10 3
FITC LOG
CHO-MQUSE MpI
CHO
urn
I I I llll
10 3
FITC LOG
FITC LOG
253
JP 2004-071763
[Fig. 9]
2
1.8
1 1.6
m
5° 1.4
10
1.2 h
1
LU
o 0.8
m 0.6
cr
g 0.4
tn
< 0.2
0
0.000
[Fig. 10]
0.001
0.010
0.100
1.000 10.000 100.000
CONCENTRATION [nM]
2
_ 1.8
I 1.6
to
$° 1.4
1.2
1
o 0.8
m 0.6
a:
S 0.4
CD
< 0.2
0
-h- AB324 DIABODY
AB317 DIABODY
■e-TPO
0.000
0.001
0.010
0.100
1.000 10.000 100.000
CONCENTRATION [nM]
254
JP 2004-071763
[Fig. 11]
0.000 0.001 0.010 0,100 1.000 10.000 100.000
CONCENTRATION [nM]
[Fig. 12]
1
0.9
CONCENTRATION (ng/ml)
255
JP 2004-071763
[Fig. 13]
1.2
1
o
o
0.8
0.6
0.4
0.2
0
0.1
[Fig. 14]
1
0.9
0.8
0.7
g0.6
20.5
O0.4
0.3
0.2
0.1
0
1 .0 1 0.0 1 00.0
CONCENTRATION (ng/ml)
1 000.0
hTPO
VB08Bdb
VB45Bdb
VB033db
VB140db
VB157db
TA136db
TA136 sc(Fv)2
TA136 db
TPO
CONCENTRATION (nM)
256
JP 2004-071763
[Fig. 15]
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
in
o
O
TA136 sc(Fv)2
TA136 db
TPO
CONCENTRATION (nM)
[Fig. 16]
0.8
0.7
0.6
Q
O
0.5
0.4
0.3
0.2
0.1
0
TA136 sc(Fv)2
TA136 db
TPO
CONCENTRATION (nM)
257
JP 2004-071763
[Fig. 17]
2
1.8
1.6
1.4
o 1.2
in
S 1
° 0.8
0.6
0.4
0.2
0
TA136 sc(Fv)2
TA136 db
TPO
CONCENTRATION (nM)
258
JP 2004-071763
259
JP 2004-071763
[Fig. 19]
o 1 1
0.000 0.001 0.010 0.100 1.000 10.000
CONCENTRATION [nM]
260
JP 2004-071763
[Document Name] Abstract
[Abstract]
[Problems to be Solved] An objective of the present invention is to provide novel anti-Mpl
5 antibodies having TPO-agonistic activity.
[Means for Solving the Problems] Anti-human Mpl antibodies were isolated and purified, and
then anti-human Mpl diabodies and anti-human Mpl sv(Fv) 2 were purified using genetic
engineering techniques. Furthermore, the present inventors succeeded in humanizing
anti-human Mpl sc(Fv)2.
10 The diabodies and sc(Fv)2 were assayed for TPO-like agonistic activity, and were found
to have activities higher than those of anti-human Mpl antibodies, or activities equivalent to or
higher than those of naturally-occurring human TPO ligand.
[Selected Drawings] None
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